HK1199070B - Methods of amplifying whole genome of a single cell - Google Patents

Description

Single-cell whole genome amplification method

Related application number

This application claims priority to the following U.S. provisional patents: #61/621,271, filed April 6, 2012; #61/550,667, filed October 24, 2011; #61/510,539, filed July 22, 2011; #61/490,790, filed May 27, 2011;

Government Interest Statement

This invention was supported by NIH Grants 3R01HG005097-02 and 3R01HG005097-02S1. The government has certain rights in this invention.

Technical Field

Embodiments of the present invention generally relate to methods and compositions for amplifying genomic sequences, such as single cell whole genome amplification.

Background Art

A common problem with random primer amplification of DNA and RNA is the formation of primer dimers. Primer concentrations must be high enough for efficient amplification. High primer concentrations lead to dimer formation, significantly reducing primer binding to the DNA or RNA template. Other issues with random primer amplification include inability to amplify the entire genome due to site loss, the generation of short amplicons, and the inability to amplify partially degraded or artificially modified DNA.

There are reports on genomic PCR amplification methods in the literature, such as US7,718,403, US2003/0108870 and US7,402,386. PCR amplification methods such as Genomeplex and Picoplex have significant bias (heterogeneity) towards different gene loci, resulting in the loss of some loci after amplification. Cell products amplified by these methods often only sequence 5-30% of the genomic sequence. Multiple displacement amplification (MDA) uses low-temperature amplification, which can lead to the formation of a large amount of chimeras. These non-genomic sequences cause significant false-positive sequencing results and sequencing artifacts. Although whole genome amplification has been attempted, these methods are generally inefficient, complex and expensive. Therefore, there is a need to develop an effective method for amplifying the entire genome from a very small amount of DNA, such as a single cell.

Summary of the Invention

This patent relates to a DNA amplification method that uses a small amount of genomic DNA, or a small amount of genomic DNA sequence, from a single cell, or a small number of cells of a single cell, or from blood or body fluids, from a human or other species. This amplification method can be performed using a single reaction tube on a PCR amplifier. This method can be used for high-throughput sequencing of whole-genomic DNA amplified from a single cell, achieving substantial sequencing depth.

This patented method amplifies various sites across the entire genome of a single cell with high fidelity, uniformity, and sequencing breadth, and the amplified products are used for high-throughput sequencing analysis. This method reduces sequencing bias, thereby providing relatively comprehensive, over 90% genomic DNA sequence information from a single cell. At sequencing depths of 7x, 10x, or 15x, over 70% of the genomic sequence information can be measured, with virtually no influence from chimera formation. This method avoids sequencing artifacts and advances advanced genomic analyses such as single nucleotide polymorphisms (SNPs), gene copy number polymorphisms (CNVs), and gene structural variations (SVs), as well as high-throughput gene sequencing analysis. This method is particularly useful for biological systems with multiple cell populations, such as tumors and neural tissue.

The primers used in this patented method contain a common sequence, a variable sequence, and a fixed sequence. After the DNA is denatured, the single-stranded primer binds to the single-stranded template at low temperature and then amplifies at high temperature using at least one polymerase with strand displacement properties and 3' end nucleic acid exonuclease activity. At the end of the second amplification cycle, the double-stranded DNA (dsDNA), with the primer sequence bound at one end and the complementary primer sequence bound at the other end, denatures to produce single-stranded DNA (ssDNA). The reaction temperature is lowered to a temperature that allows the free primer to hybridize with the 3' end of the single-stranded amplicon, thereby preventing the amplicon from hybridizing with itself or with each other, and then cooled to an appropriate temperature to complete the primer binding and the next amplification cycle.

This patented method can be used to amplify small or trace amounts of DNA, perform karyotyping or high-throughput screening on multiple locations within a sample DNA sample, and rapidly establish probes specific for specific chromosomal regions, perform microdissection, or amplify marker chromosomes of unknown chromosomal regions or labeled abnormal chromosomes. It can also be used for rapid cloning of amplifications or the establishment of DNA libraries. Therefore, this method is not only useful for karyotyping and high-throughput screening, but also valuable for cytogenetic diagnosis.

This patented method uses an unconventional PCR cycle amplification method to amplify whole genome sequences from single mammalian cells. Certain DNA polymerases can produce typical results. An additional amplification step allows free primers to bind to complementary sequences on the amplicon, such as the 3' end. This prevents hybridization of the amplicon to itself or to each other, leading to the formation of chimeras, and provides a method for removing primers from both ends of the amplicon.

This patented method amplifies the whole genome sequence of one or several cells. In the usage diagram, a single cell is first separated and lysed in a certain volume of cell lysis solution to release genomic DNA. The lysate can be divided into 2 to 100,000 subparts. The DNA separation method can divide two homologous gene sites into different subparts. The more subparts there are, the higher the possibility that the homologous DNA sites are divided into different subparts. The two sites of homologous DNA are divided into different subparts, which can be used for karyotype analysis of genomic DNA hemiploidy. The genomic components in different subparts can be amplified to obtain the corresponding part of the genomic amplification products for sequencing analysis. Two or more samples of the same or different cells can be processed and analyzed as single cell samples.

This patented method amplifies the whole genome sequence of one or several cells. The genetic material of a single cell can be divided into several subfractions and amplified and analyzed separately, enabling haploid or diploid karyotyping. This method can be used to sequence and assemble de novo genomes from species for which no reference genome sequence exists or whose genomes exhibit complex structural variation. The method can be used with DNA from diverse sources, including heterogeneous tissues such as tumors, rare and precious samples such as embryonic stem cells, and non-dividing cells such as neurons. It can also be used with different sequencing platforms and karyotyping methods. Haploid karyotype analysis methods can be found in the following literature: Levy, S. et al. The diploid genome sequence of an individual human. PlosBiol.5, e254 (2007); de Bakker, P. I. et al. A high-resolution HLA and SNP haplotypemap for disease association studies in the extended human MHC. Nat. Genet. 38, 1166-1172 (2006); Nagel, R. L. et al. The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients. haplotypes alter receptor expression and predict in vivo responsiveness.Proc.Natl.Acad.Sci.USA97, 10483-10488(2000); Sun, T.et al.Haplotypes in matrix metalloproteinase gene cluster onchromosome11q22contribute to the risk of lung cancer development and progression.Clin.Cancer Res.12, 7009-7017(2008); Kitzman, J.O.et al.Haplotype-resolved genome sequencing of a Gujarati Indian individual.Nat.Biotech.29, 59-63(2011); International HapMap Consortium.Integrating common and rare genetic variation in diverse human populations.Nature467, 52-58(2010); Xiao, M.et.Al.Direct determination of haplotypes from single DNAmolecules. Nat. Methods 6, 199-201 (2009); Fan H.C. et al. Whole-genome molecular haplotyping of single cells. Nat. Biotech. 29, 51-57 (2011).

Other relevant features and advantages of the patented method are more clearly explained in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The above-mentioned method of this patent and other related characteristics and advantages are clearly explained in the following usage diagrams.

Figure 1: Schematic diagram of single-cell DNA amplification

Figure 2: Single-cell genome sequencing results, sequencing depth 15x.

Figure 2A: Single-cell genome sequencing results for all chromosomes 1.

Figure 3: Flowchart of single-cell DNA amplification for high-throughput sequencing, including linear pre-amplification and exponential amplification stages.

Figure 4: Schematic diagram of the patented method, Multiple Annealing Loop Amplification Cycles (MALBAC). First, a primer binds to a single-stranded DNA template and is extended by a polymerase with strand-displacing activity, producing a semi-amplifier. Next, a single-stranded fully amplified product with complementary sequences at both ends is generated. This fully amplified product self-ligates at an intermediate temperature, rendering it ineffective as a template at this step. This results in near-linear amplification, avoiding common amplification bias.

Figure 5A is a Lorenz curve comparing the uniformity of sequencing breadth for MALBAC, MDA, and unamplified samples. Single-cell and unamplified samples are from the SW480 cell line. Data for the MDA method are from Fan et al., Nat. Biotech. 29, 519 (2011). Completely uniform and unbiased amplification should appear as a diagonal line in the graph, with the greater the deviation from the diagonal line, the more severe the amplification bias. The arrows represent the portion of the genome that was not amplified (MDA 55%, MALBACl 5%, unamplified sample 5%). Figure 5B is a whole-genome read distribution diagram. The results for MALBAC and unamplified samples are similar, while the MDA method has a large number of low-density regions, representing regions of several million bases that are underestimated or overestimated in the amplified product.

DETAILED DESCRIPTION

This patent involves general conventional operating methods, such as molecular biology techniques, recombinant DNA techniques, and microbiology techniques, which have been described in detail in the literature. See: Sambrook, Fritsch, and Maniatis, Molecular Cloning: A laboratory Manual, ^2nd Edition (1989), Oligonucleotide synthesis (MJGait, Ed., 1984), Animal Cell Culture (RIFreshney, Ed., 1987), Methods in Enzymology (Academic Press, Inc Series, Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos) eds, 1987), Handbook of Experimental Immunology (DMWeir and CCBlackwell, Eds), Current Protocols in Molecular Biology (FMAusubel et al eds., 1987), Current Protocols in Immunology (JEColigan etal eds., 1991), Annual Review of Immunology, Advances in Immunololgy.

Terms and symbols used in nucleic acid chemistry, biochemistry, genetics, and molecular biology follow standard professional usage, such as Komberg and Baker, DNA Replication, ^2nd Edition (WH Freeman, NY, 1992); Lehninger, Biochemistry, ^2nd Edition (Worth Publishers, NY, 1975); Strachan and Read, Human Molecular Genetics, ^2nd Edition (Wiley-Liss, NY, 1999); Eckstein, editor, Oligonucleotides and analogs: A practical approach (Oxford University Press, NY, 1991); and Gait, editor, Oligonucleotide synthesis: A practical approach (IRL Press, Oxford, 1984).

The invention is based in part on the establishment of a method for amplifying DNA or RNA (such as mRNA) using genomic or transcriptomic material or both. This DNA or RNA may come from a single cell, or a few cells of the same cell. The method can be used to amplify the DNA of any species or organism, and can be used in an amplification reaction in a PCR tube. The DNA can also be divided into several sub-portions, using a pipette or other micro-pipetting device so that each sub-portion contains only part of the original DNA, which may represent genomic DNA at the subcellular level, that is, part of the DNA of a single cell genome, and is amplified and analyzed accordingly. The method can be used for DNA amplification that does not rely on a specific DNA sequence, and the DNA can come from but is not limited to human, animal, plant, fungal, viral or other eukaryotic or prokaryotic cell DNA.

This inventive method can be used to amplify nearly all genes in a genome or transcriptome (respectively, termed whole-genome amplification and whole-transcriptome amplification) without losing the representativeness of the amplified sequence due to missing specific regions. "Nearly the entire genome" refers to sequences encompassing at least 80-99% of the genome. This amplification method can sometimes result in unequal amplification of sequences in different parts of the genome.

The method of the invention can be carried out in two steps in a single reaction tube or microplate. In the first step, an amplification library is generated, and the primers used contain a common sequence, a variable sequence and a fixed sequence, and at least one polymerase with chain displacement activity or nucleic acid exonuclease activity. The method includes a post-annealing step, which allows the free primers to bind to the ends of the amplification products, thereby preventing the amplification products from binding to themselves or each other. This linear amplification can significantly reduce the problem of amplification bias. The specific primer design and post-annealing step maximize the amplification breadth of the genome. In the second step, the molecules in the amplification library are amplified by standard PCR, using Taq polymerase and primers for known sequences, thereby performing unbiased amplification of the entire genome or the entire transcriptome by thousands of times, and the products can be further amplified to more than millions of times.

This method uses at least one DNA polymerase to amplify DNA. Primers bind to a DNA template, and the polymerase synthesizes a complementary strand starting from the 3' end of the primer. The specific steps can be divided into denaturation of the template DNA, binding of the primer to the template DNA, and extension of the primer by the polymerase on the template to synthesize the complementary strand.

Generally speaking, DNA amplification begins with the high-temperature denaturation of a dsDNA template into ssDNA. The temperature is then lowered (annealed), allowing primers to bind to the ssDNA template. One or more polymerases in the system then extend the primers along the template, synthesizing a complementary strand. DNA polymerases may contain 5' to 3' exonuclease activity or strand displacement activity.

After the first amplification cycle, the 5' end of the newly synthesized complementary chain carries the primer sequence. The above denaturation, annealing, and extension steps are repeated.

After each cycle, the amplified DNA molecule has the primer sequence at one end and the primer's complementary sequence at the other end. When the reaction conditions are appropriate, the free primer can bind to the template, thus preventing the amplicons from binding to themselves or to each other.

The DNA template can be genomic DNA, microdissected chromosomal DNA, YAC, cosmid DNA, phage DNA, PAC DNA, BAC DNA; can be from mammals, plants, fungi, viruses, or prokaryotic DNA; can be from humans, cows, dogs, pigs, mice, birds, fish, shrimp, plants, fungi, viruses, or bacteria.

The primers used in the inventive method include a common (constant) sequence, a variable sequence and a fixed sequence, sometimes also referred to as quasi-degenerate primers. The common sequence and the variable sequence contain sequences that are essentially not complementary to themselves or other primers in the reaction. The common sequence is preferably known and can be the amplification target sequence. The variable sequence can be selected randomly or selected accordingly based on the source of the template DNA and the sequence characteristics. The common sequence is common to all primers in the reaction, is 10-30bp long, and does not contain C. The variable sequence is 3-7bp long and can contain any base. If it is 5 bases long, there can be 4 to the power of 5 sequence combinations. The fixed sequence is 2-4bp long and does not contain complementary sequences, such as GGG, TTT, GAA, and ATG.

Typical primers include 5'-GT GAG TGA TGG TTG AGG TAG TGT GGAGNNNNNGGG-3' and 5'-GT GAG TGA TGG TTG AGG TAG TGTGGA GNNNNNTTT-3'. The 5N3G and 5N3T pair can also be replaced by the following pairs: 5N3G and 5N3A; 5N3C and 5N3T; and 5N3C and 5N3A. The consensus sequence can be modified to reduce primer-dimer formation.

The reaction mixture contains a denatured single-stranded template and primers. The primers can be one or more degenerate primers that bind to at least a certain portion of the single-stranded template molecule. The template can be DNA or RNA.

The reaction mixture contains primers, a template, and at least one DNA polymerase with 5' to 3' exonuclease activity or strand displacement activity. The DNA polymerase with strand displacement activity can open the double-stranded DNA template during extension, and includes polymerase, Bst polymerase, pyrophage 3173, vent polymerase, deep vent polymerase, TOPO Taq, vent (exo-) polymerase, deep vent (exo-) polymerase, 9°Nm polymerase, DNA polymerase Klenow fragment, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, a variant of T7 bacteriophage DNA polymerase (without exonuclease activity), or any mixture thereof. The one or more polymerases contain 5' to 3' exonuclease activity, such as Taq polymerase, Bst DNA polymerase (full length), Escherichia coli DNA polymerase, LongAmp Taq polymerase, OneTaq polymerase, or any mixture thereof.

The mixed reactants undergo multiple amplification cycles. The annealing temperature is below 30°C, and the extension temperature can be above 10-65°C depending on the type of polymerase. Polymerase activity is best above 30°C, while Bst polymerase and pyrophage3173 (exo-) are most active above 62°C. The denaturation temperature is between 90-100°C. There is also an additional annealing temperature of 55-60°C, with 58°C generally used. After the denatured amplicon is denatured, the mixture binds to its own ends at this temperature and temporarily stops serving as a template, thus achieving linear amplification.

The amplicon can be processed to remove the two terminal primer sequences, or further subjected to conventional PCR amplification, or directly subjected to high-throughput sequencing analysis if the quantity is sufficient.

The DNA denaturation temperature is 90-100°C, typically 95°C, and the denaturation time is 10 seconds to 5 minutes, typically 2 minutes.

Annealing temperature is 0-30°C, annealing time is 10 seconds to 5 minutes. The extension temperature depends on the DNA polymerase used. When multiple polymerases are present, a temperature gradient can be used to activate different enzyme activities. The extension time is 2-7 minutes, typically 5 minutes.

The above parameters such as temperature and time can be changed appropriately and generally will not significantly affect the amplification effect.

As shown in Figure 3, the separated single cells are lysed to release genomic DNA, which is then linearly pre-amplified and then subjected to conventional PCR amplification. The final product is used for high-throughput sequencing or screening.

As shown in Figures 1, 3, and 4, the single-cell genome amplification conditions of MALBAC (Multiple Annealing and Looping) technology begin with a linear pre-amplification process, which is used to amplify repetitive fragments that cover the majority of the genome. This pre-amplification is followed by traditional exponential amplification, such as PCR, to further amplify the genome. The amplicon can then be used for next-generation sequencing.

As shown in Figures 1, 3, and 4, the nucleotides of a single cell, such as a chromosome, serve as the template in the amplification system. The nucleotides of the single cell are denatured into single strands and serve as the template for amplification. Under suitable conditions, a large number of random primers hybridize with the single-stranded nucleotides, and under the action of a polymerase with strand displacement activity, a complementary fragment of the single-stranded nucleic acid template is synthesized. The random primers consist of a fixed base sequence and a degenerate base sequence. The amplification process begins at a random site within the single-stranded nucleic acid template. The nucleic acid fragments amplified using the single-stranded nucleic acid template are called first-round amplicons or semi-amplicons.

Complementary sequences hybridizing to single-stranded nucleic acid templates can be denatured into single strands under appropriate conditions, maximizing the amount of single-stranded nucleic acid template available for hybridization with primers. Double-stranded nucleic acids in the amplification mixture are denatured into single strands by the action of strand-displacing polymerases, increasing the amount of single-stranded nucleic acid available for hybridization with primers and amplicon.

The first round of amplification, also known as the half-amplification, has complementary 5' and 3' ends, allowing it to form a circular structure, known as a semi-amplification ring. The amplification efficiency of the semi-amplification ring is very low, and the semi-amplification ring prevents further amplification, which is an important step in linear amplification.

The linear first-round amplicon is present in the amplification mixture along with the single-stranded nucleic acid. The first-round amplicon and the single-stranded nucleic acid serve as primer templates in the second-round reaction.

Under appropriate conditions, the primers hybridize to different positions on the first round amplicon and the nucleotide single strand.

Under suitable conditions, a polymerase with strand displacement activity acts on the binding site between the first-round amplicons and random primers, amplifying the complementary sequences of each first-round amplicons. The ends of these amplified complementary sequences have the complementary sequences of the primers. The products amplified using the first-round amplicons as templates are the second-round amplicons or full amplicons. Similarly, a polymerase with strand displacement activity also acts on the binding site between a single-stranded nucleic acid and random primers, amplifying the complementary sequences of the single-stranded nucleic acid. The products amplified using a single-stranded nucleic acid as a template are the first-round amplicons. As mentioned above, some first-round amplicons will form circular half-amplicons.

The complementary fragments hybridize to nucleic acids in the amplification mixture, such as single-stranded template nucleic acid, half-amplid, or full-amplid, through an annealing step. Under appropriate conditions, the nucleic acid sequences in the amplification mixture are denatured into single strands, which can increase the amount of single-stranded nucleic acids available for hybridization with the primers and amplification product. Despite the use of a polymerase with strand displacement activity, the amplification system still contains double-stranded nucleic acids, which can also be denatured into single strands, increasing the amount of single-stranded nucleic acids available for hybridization with the primers and amplification product.

In order to reduce the formation of polymers caused by the 3' end of the full amplification product binding to the 5' end of another full amplification product or other nucleotides, the two ends of the second round of amplification products are complementary and hybridized into a ring structure, so that the second round of amplification products cannot form polymers and cannot serve as a template for amplification. Therefore, under suitable conditions, excess primers hybridize with the 3' end of the second round of amplification products, and amplification appears in a linear pattern without polymer production. This patent uses the 5' and 3' ends of the full amplification products to complement each other into a ring to reduce the production of polymers, etc., and prevent the full amplification product from serving as a template for the next round of amplification cycles. At the same time, the DNA and half-amplification products in the amplification mixture system can serve as templates for the next round of amplification cycles.

During the subsequent cycles, under appropriate conditions, the primers hybridize to and amplify the first-round amplification product and the nucleic acid single strand. However, the second-round amplification product or the full amplification product may be unable to proceed to the next amplification step due to the 3' end of the primer binding or the 5' and 3' ends hybridizing to form a loop. The amplification mixture is then placed under conditions suitable for amplifying the nucleic acid single strand and the half amplification product into a half amplification product and a full amplification product, followed by conditions suitable for the second-round amplification product or the full amplification product to hybridize into a loop. The cycle is repeated as needed.

Further analysis methods of the amplified DNA products include genotyping analysis of the DNA amplification products. In addition, the amplified DNA products can be used to identify polymorphisms of the DNA amplification products, such as single nucleotide polymorphism analysis (SNP). In the experimental protocol, SNPs can be detected by some well-known methods, including DNA sequencing, by amplifying a PCR product and then sequencing; oligonucleotide ligation assay (OLA), single base extension method, allele-specific primer extension method, mismatch hybridization method. The discovered SNPs are preferably shown to be associated with phenotypes, including disease phenotypes. DNA libraries can be constructed from DNA amplification products, including non-restricted genomic DNA libraries, microchromosomal DNA libraries, BAC libraries, YAC libraries, PAC libraries, cDNA libraries, phage libraries and plasmid libraries.

The term "genome" is defined as all genes in an individual, cell, or organelle. The term "genetic DNA" is defined as all DNA in an individual, cell, or organelle. The term "transcriptome" is defined as the RNA of a single cell.

The term "nucleoside" refers to a molecule composed of a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Nucleosides include adenine, guanine, cytidine, uridine, and thymidine. Nucleosides also include rare nucleosides such as inosine, methylinosine, pseudouridine, 5,6-dihydrouridine, thymidine ribonucleotide, ^2N -methylguanine, and ^2,2N ,N-dimethylguanine. The term "nucleotide" refers to a molecule composed of a nucleoside and one or more phosphate groups. Nucleotides include adenine, guanine, cytosine, and thymidine. The terms "polynucleotide,""oligonucleotide," and "nucleotide molecule" all refer to polymers of nucleotides, macromolecules composed of deoxynucleotides or ribonucleotides linked by 3',5'-phosphodiester bonds. Polynucleotides have various three-dimensional structures and numerous functions. The following are all polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), exon, intron, messenger RNA (mRNA), transfer RNA, ribonucleic acid (RNA), ribozyme cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, DNA sequence, RNA sequence, nucleotide probe and primer. A polynucleotide molecule is composed of modified nucleotides, such as methylated nucleotides and nucleotide analogs. Unless otherwise stated, the polynucleotides of this patent have a double-stranded structure and complementary single-stranded structures. A polynucleotide is a special sequence composed of four nucleotide bases, including adenine (A), thymine (T), guanine (G), cytosine (C); when the polynucleotide is RNA, it has uracil (U). Therefore, the polynucleotide sequence is represented by the base letters of the polynucleotide molecule. These letter sequences can be recorded in a computer database and can be analyzed using bioinformatics software for applications such as functional genomics and homology analysis.

The terms "RNA,""RNAmolecule," and "ribonucleic acid molecule" are composed of ribonucleotides. The terms "DNA,""DNAmolecule," and "deoxyribonucleotide molecule" are composed of deoxyribonucleotides. Both DNA and RNA are synthesized naturally (e.g., by DNA replication and transcription, respectively). DNA and RNA can exist as single strands (e.g., ssRNA and ssDNA) or multiple strands (e.g., dsDNA and dsRNA). "mRNA" or "messenger RNA" is single-stranded RNA that determines the sequence of amino acids in a peptide chain .

In this patent, the term "small interfering RNA"("siRNA") (also referred to as "short interfering RNA" in the literature) refers to an RNA (or RNA analog) of 10 to 50 nucleotides (or nucleotide analogs) in length that can guide or mediate RNA interference. An siRNA can be between 15 and 30 nucleotides or nucleotide analogs, between 16 and 25 nucleotides or nucleotide analogs, between 18 and 23 nucleotides or nucleotide analogs, or even between 19 and 22 nucleotides or nucleotide analogs (i.e., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to an siRNA composed of 21 nucleotides (or nucleotide analogs), such as 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers to an siRNA composed of about 24 to 25 nucleotides, such as 23, 24, 25 or 26 nucleotides. Short siRNAs may include shorter siRNAs of less than 19 nucleotides, such as 16, 17 or 18 nucleotides, that have the ability to mediate RNAi. Long siRNAs may include longer siRNAs of more than 26 nucleotides that have the ability to mediate RNAi and are not further processed, such as enzymatically cleaved into short _siRNAs .

The terms "nucleotide analogs," "alternative nucleotides," and "modified nucleotides" refer to non-standard nucleotides, including non-naturally occurring nucleotides or deoxynucleotides. Nucleotide analogs are nucleotides that have been modified at any position to alter specific chemical properties while still retaining the ability to perform the desired function as a nucleotide analog. Examples include derivatives of nucleotides at the 5-position (e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propynyluridine, 5-propyleneuridine), the 6-position (e.g., 6-(2-amino)propyluridine), and the 8-position (e.g., 8-propynyluridine, 8-chloroguanosine, 8-fluoroguanosine). Nucleotide analogs also include deaza nucleotides, such as 7-deaza-adenosine; O- and N-modified (e.g., alkylated, such as N6-methyladenosine or other modifications known in the literature) nucleotides; or other heterocyclic modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4): 297-310.

Nucleic acid analogs may also be produced by modifying the sugar moiety in nucleotides. For example, the 2'OH group may be substituted with H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, COOR, or OR, where R may be substituted or unsubstituted with C1-C6 alkyl, alkenyl, alkynyl, or aryl. Other possible modifications include those described in U.S. Patent Nos. 5,858,988 and 6,291,438.

The phosphate groups of nucleotides may also be modified, for example, by replacing one or more oxygen atoms on the phosphate group with sulfur (e.g., phosphorothioate), or by other substitutions to enable the nucleotide to perform the desired function, as described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21; Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5): 333-45; Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25; Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2): 77-85 and U.S. Patent No. 5,684,143. The above modifications (e.g., phosphate group modifications) will reduce the hybridization efficiency of nucleotide analogs in in vivo and in vitro assays.

In this patent, the term "isolated RNA" (i.e., "isolated mRNA") refers to the complete separation of RNA molecules from other cellular materials or culture medium when using recombinant technology; or the complete separation of chemical precursors or other chemicals when using synthetic technology.

The term "in vitro" refers to the use of purification reagents or extraction reagents, or in environments such as cell extraction. The term "in vivo" refers to the use of living cells, that is, immortalized cells, primary cells, cell lines, and/or cells within an organism.

The terms "complementary" and "complementarity" are used to describe nucleic acid sequences with respect to the base pairing rules. For example, the sequence 5'-AGT-3' is complementary to the sequence 5'-ACT-3'. Complementarity can be partial or complete. Partial complementarity occurs when one or more nucleic acid bases cannot be matched according to the base pairing rules. Complete or total complementarity occurs when every base in one nucleic acid matches every base in the other. The degree of complementarity between nucleic acid chains strongly influences the efficiency and strength of hybridization.

The term "homology" when referring to a relationship between nucleic acids refers to the degree of complementarity. There can be partial homology (i.e., partial identity) as well as complete homology (i.e., complete identity). A partially complementary sequence is one that can at least partially inhibit the hybridization of a completely complementary sequence to a target nucleic acid, and is related to the functional term "high identity." Inhibition of hybridization of a completely complementary sequence to a target sequence can be detected under low stringency conditions in hybridization assays (e.g., Southern or Northern blotting, or liquid phase hybridization). A highly homologous sequence or probe (i.e., an oligonucleotide capable of hybridizing to a target oligonucleotide) can compete with and inhibit the binding (i.e., hybridization) of a completely homologous sequence to the target under low stringency conditions. This does not mean that low stringency conditions will result in nonspecific binding; low stringency conditions require that the two sequences that bind to each other interact specifically (i.e., selectively). The presence of nonspecific binding can be detected by using a second target that is not partially complementary (i.e., less than 30% identical); a probe that does not exhibit nonspecific binding will not hybridize to a non-complementary target.

When the term "highly homologous" is applied to double-stranded nucleic acids such as cDNA or genomic clones, it means that any probe can hybridize to one or both strands of the double-stranded nucleic acid under low stringency conditions. When the term "highly homologous" is applied to single nucleotide chains, it means that any probe can hybridize to the single-stranded nucleic acid sequence under low stringency conditions.

The following terms are used to describe the relationship between two or more sequences: "reference sequence," "sequence identity," "percentage of identical sequences," and "highest identity." A "reference sequence" is a basis for sequence comparison; a reference sequence may be a portion of a longer sequence, such as a portion of a full-length cDNA in a given sequence listing or consist of a complete gene sequence. Typically, a reference sequence is 20 nucleotides long, often at least 25 nucleotides long, and more often 50 or more nucleotides long. Because two polynucleotides may (1) have a sequence (i.e., a portion of the complete polynucleotide sequence) that is similar in both polynucleotide sequences and (2) may also differ in other regions, a typical approach to comparing two (or more) sequences is to compare the two sequences over a "comparison window" and identify the sequence identity within that region. The "comparison window" here refers to a conceptually shortest segment of 20 consecutive nucleotide positions that may match at least 20 consecutive nucleotides in the reference sequence. The polynucleotides in the comparison window may have 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions). The best match between sequences within the alignment window can be obtained by using a computer to run the Smith and Waterman local homology algorithm (Smith and Waterman, (1981) Adv. Appl. Math. 2: 482), the Needleman and Wunsch (J. Mol. Biol. 48: 443 (1970)) local homology algorithm, the Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444 (1988)) similarity search method and other algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Version 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by running multiple methods and selecting the best match (i.e., the result with the highest proportion of identity in the alignment window).

The term "sequence identity" refers to two polynucleotide sequences that are identical (i.e., form nucleotide-nucleotide pairs) within the alignment region. The term "percentage of identical sequences" is calculated by comparing matching segments of the two sequences to the alignment region, determining the number of identical nucleic acid base positions (i.e., A, T, C, G, U, or I) in the two sequences, dividing the number of identical nucleic acid positions by the total number of positions within the comparison region (i.e., the alignment window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The term "highly homologous" is used to describe a polynucleotide sequence that is at least 85% identical to another sequence, preferably having a sequence identity of 90-95%, and more generally refers to a sequence that is 99% or more identical to a reference sequence over an alignment region of 20 or more nucleotides, typically 25-50 nucleotides. The reference sequence may be a portion of a longer sequence, such as a fragment of the full-length sequence claimed in the present invention.

The term "hybridization" refers to the pairing of complementary nucleic acids. Hybridization and its strength (i.e., the strength of the binding between nucleic acids) are influenced by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions, the Tm of the hybridization, and the G:C ratio of the nucleic acids.

The term "Tm" refers to the melting temperature of nucleic acids. The melting temperature is the temperature at which half of a double-stranded nucleic acid molecule dissociates into single strands. For nucleic acid in a 1M NaCl aqueous solution, the Tm value can be simply estimated using the following formula: Tm = 81.5 + 0.41 (% G + C) (see, for example, Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985)). Some more complex Tm calculation methods take both nucleic acid structure and sequence characteristics into account.

The term "stringency" refers to the conditions during nucleic acid hybridization, such as temperature, ionic strength, and the presence of other substances such as organic solvents.

In nucleic acid hybridization, "low stringency conditions" refer to binding or hybridization at 42°C, a hybridization solution containing 5×SSPE (43.8 g/l NaCl, 6.9 g/l _NaH2PO4 ( _H2O ) and 1.85 g/l _EDTA , pH adjusted to 7.4 with NaOH), 0.1% SDS, 5×Denhardt's reagent (each 500 ml of 50×Denhardt's reagent contains: 5 g Ficoll (Type 400; Pharmacia), 5 g BSA (Fraction V; Sigma)) and 100 μg/ml denatured salmon sperm DNA, followed by washing with 5.0×SSPE (containing 0.1% SDS) solution at 42°C (if the probe is approximately 500 nucleic acids in length).

In nucleic _acid hybridization, "moderate stringency conditions" refer to binding or hybridization at 42°C, a hybridization solution containing 5×SSPE (43.8 g/l NaCl, 6.9 g/l _NaH2PO4 ( _H2O ) and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA, followed by washing with 1.0×SSPE (containing 1.0% SDS) solution at 42°C (if the probe is approximately 500 nucleic acids in length).

"High stringency reaction conditions" refer to incubating the hybridization probe (approximately 500 nt) at 42°C with a mixture of _{5 ×} SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4 ( _H2O ), 1.85 g/l EDTA, and adjusted to pH 7.4 with NaOH), 0.5% SDS, 5×Denhardt's reagent, and 100 μg/ml denatured salmon sperm DNA; after the probe binds to the nucleic acid, elution is performed at 42°C with an elution buffer (0.1×SSPE, 1.0% SDS).

Many factors can influence reaction stringency. These factors include probe length, type, and base composition; target molecule type, base composition, and storage conditions; the concentrations of salt ions and other components (the presence or absence of formamide, dextran sulfate, and polyethylene glycol); and hybridization conditions. These factors can all contribute to low reaction specificity. Furthermore, high stringency can be achieved by increasing the hybridization temperature or adding formamide to the eluent.

In some embodiments, cells are first identified, followed by isolation of single cells or multiple cells. The cells referred to in this patent include all cells whose DNA or RNA content is recognized. This includes various types of cancer cells, such as hepatocytes, oocytes, embryos, stem cells, iPS cells, ES cells, neurons, red blood cells, melanocytes, astrocytes, germ cells, oligodendrocytes, kidney cells, etc. The method in this patent is successfully applied to the DNA or RNA of single cells. Multi-cells include from about 2 to 1,000,000 cells.

The nucleic acid referred to in the present method can be DNA, RNA, or a DNA-RNA chimera. The nucleic acid can be obtained from a variety of sources, such as human samples including whole blood, serum, plasma, cerebrospinal fluid, facial epidermal cells, breast milk, biopsy tissue sections, semen, urine, feces, hair follicles, saliva, sweat, immunoprecipitated or physically isolated chromatin, etc.

The nucleic acid molecules obtained by the present method, amplified from the template, can provide diagnostic or prognostic information. For example, the processed nucleic acid molecules obtained from the sample can provide genomic copy and/or sequence information, allelic variation information, cancer diagnosis, prenatal diagnosis, paternity testing, disease diagnosis, detection, monitoring, and corresponding treatment information, sequence information, and the like.

The "single cell" referred to in this article means "single cell". The sample source of the single cell can be tissue sections, whole blood or cell culture fluid. Cells derived from specific organs, tissues, tumors, etc. are also suitable for this method. In addition, cells of prokaryotic (such as bacteria) or eukaryotic (such as yeast) microbial populations are also applicable to this method. Single-cell suspensions can be obtained by conventional methods, for example, using trypsin or papain to enzymatically hydrolyze the sample tissue or using physical methods to separate cells. The obtained single cells can be stored in different sterile centrifuge tubes for separate processing, such as a 96-well plate, so that one single cell can be placed in each well.

Currently, commonly used methods for manipulating single cells include fluorescence-activated cell sorting (FACS), flow cytometry (FCM; Herzenberg, PNAS USA 76:1453-55, 1979), micromanipulation, and semi-automated cell harvesting (e.g., the Quixell ^™ cell transfer system from Stoelting). Single cells can be selected and identified by microscopic observation of location, morphology, or marker gene expression. Furthermore, combining gradient centrifugation with flow cytometry can improve the efficiency of cell separation and screening.

Once the desired cells are identified, technicians can use common methods to lyse the cells and release intracellular materials, including DNA and RNA, all in a test tube. Methods for lysing cells include using heat, detergents, other chemical methods, or a combination of these methods. Gentle cell lysis helps prevent the release of nuclear chromatin. For example, incubating cells in Tween-20 at 72°C for 2 minutes is sufficient to fully lyse the cells, but it will cause degradation of the nuclear chromatin genome. Therefore, the following methods can be used for cell lysis: 10 minutes in a 65°C water bath (Esumi et al., Neurosci Res., 60(4): 439-51, 2008); or incubating PCR buffer II (Applied Biosystem) with 0.5% NP-40 at 70°C for 90 seconds; or using proteinase K for enzymatic digestion; or using guanidinium isothiocyanate solution (U.S. Patent No. 2007/0281313). Genomic DNA can be amplified directly from the cell lysate, that is, the reaction mixture can be directly added to the lysate. Alternatively, the cell lysate can be divided into multiple tubes so that each reaction tube contains genomic DNA and then amplified separately.

The nucleic acids used in the present invention include both natural bases and non-natural bases. Natural nucleic acids refer to one or more of adenine, thymine (uracil in RNA), cytosine and guanine. Nucleic acids, whether containing a natural skeleton or having a similar structure, include typical non-natural bases including but not limited to: inosine, xanthine, hypoxanthine, isocytosine, isoguanine, 5-methylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil ... Azacytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyadenine or guanine, 5-halouracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. and their analogs. Under certain conditions, isocytosine and isoguanine can be added to the nucleic acid sequence to reduce the non-specificity of hybridization, as shown in U.S. Patent No. 5,681,702.

The "primer" referred to in this article is a natural or artificially synthesized oligonucleotide chain. The primer can bind to the template nucleic acid, extend along the 3' end under the action of DNA polymerase, and finally complete the replication of the template. The primer length is generally 3-36nt, and the primer length in this patent is 17-30nt, including orthogonal primers, amplification primers, construction primers, etc. A pair of primers can bind to one or more sequences respectively. Primers and probes can be degenerate or quasi-degenerate sequences. The primers in the present invention bind to adjacent target sequences. A "primer" can be regarded as a short polynucleotide chain, whose free 3'-OH group binds to the target sequence or template through hybridization, and then performs complementary pairing with the target sequence. The primers of the present invention include nucleotides ranging from 17nt to 30nt.

This patented method uses PCR for DNA amplification. PCR, or polymerase chain reaction (U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188), involves a pair of specific primers that bind complementary to the target sequence and undergo a thermal cycling reaction (denaturation-annealing-extension) under the action of DNA polymerase, thereby obtaining a large number of intact target fragments, i.e., PCR products. The two primers each bind complementary to the double-stranded template. During amplification, the mixture first undergoes template denaturation. Then, the primers anneal to their complementary template sequences. The primers then undergo extension under the action of DNA polymerase, ultimately forming a new complementary strand. This "denaturation-annealing-extension" cycle is called a single cycle, and repeated multiple times can yield a large number of target fragments from the target sequence. The length of the amplified fragment is determined by the relative positions of the primers to the template and is therefore a controllable parameter.

Based on the PCR reaction, a variety of methods can be used to amplify and enrich single copies of the target sequence in genomic DNA to detectable levels (such as hybridization with labeled probes, biotin-labeled primers and antibiotic-enzyme complex binding detection, and primer labeling with ^32P dNTPs such as dCTP or dATP). As long as there is a suitable primer pair, in addition to genomic DNA, any oligonucleotide or polynucleotide can also serve as a template in the PCR reaction. In particular, the amplified fragments generated by the PCR method can also serve as templates for subsequent PCR amplification. The DNA polymerase used in PCR is a thermostable enzyme. In Southern and Northern experiments, primers also serve as probes for hybridization reactions.

Amplification, such as PCR, ligation-coupled recombination (LCR), and amplification. These are commonly used methods, and please refer to the following: U.S. Patent Nos. 4,683,195 and 4,683,202; Innis et al., PCR protocols: a guide to method and applications" Academic Press, Incorporated (1990) (for PCR); Wu et al. (1989) Genomics 4: 560-569 (for LCR). The general PCR process includes the following steps: (i) primers specifically bind to the DNA template; (ii) subsequent amplification under the action of DNA polymerase, involving annealing, extension, and denaturation; (iii) detection of the size of the PCR product. Primers should be specific and of appropriate length, that is, each primer should be specifically designed to complement the template according to the gene locus.

The reagents and equipment required for the amplification reaction are all commercially available. Primers used in the amplification reaction are preferably specifically complementary to the target gene sequence. The amplified product can be directly used for sequencing.

The amplified complementary or homologous chains can be quantified based on the base ratio and the principle of base complementary pairing.

"Reverse transcription PCR" or "RT-PCR" refers to the conversion of template mRNA into complementary single-stranded DNA or cDNA by the action of reverse transcriptase, followed by PCR amplification of the DNA.

The terms "PCR product," "PCR fragment," and "amplicon" all refer to the mixture resulting from a PCR reaction that has undergone two or more cycles of denaturation-annealing-extension. These terms also encompass the simultaneous amplification of one or more fragments of one or more target sequences.

The term "amplification reagents" includes reagents such as dNTPs and buffer, as well as primers, nucleic acid templates, and amplification enzymes required for amplification. Amplification reagents and other reaction components are placed in a reaction tube (test tube, centrifuge tube, etc.) for the reaction. Amplification methods include conventional PCR, rolling circle amplification (HCA), hyperbranched rolling circle amplification (HRCA), and loop-mediated isothermal amplification (LAMP).

Emulsion PCR (ePCR) involves vigorous shaking or stirring of a water-in-oil mixture to generate millions of micron-sized aqueous compartments for individual amplification. The DNA library is mixed with limiting dilution magnetic beads prior to emulsification or directly into the emulsion mixture. The size of these compartments, along with the limiting dilution magnetic beads and target molecules, form a microreactor, which contains, on average, only one DNA molecule and magnetic bead (under optimal dilution conditions, many reaction chambers contain only magnetic beads and no target molecules). To improve amplification efficiency, upstream PCR primers (low concentration) and downstream PCR primers (high concentration) are both included in the above mixed reaction solution. Depending on the size of the aqueous compartments, emulsion PCR can generate 3× ¹⁰⁹ separate PCR reactions. Depending on the emulsification state, the average particle size of the compartments in an emulsion can range from submicron to over 100 microns.

"Identity," "homology," and "similarity" are used interchangeably to refer to the sequence similarity between two nucleic acid molecules. Identity can be determined by comparing the positions of the target gene in each sequence. Homology is determined when the compared sequences contain the same base or amino acid at the same position. The degree of identity between sequences is related to the number of matches or sequences that share the same position. Sequence identities between unrelated or non-homologous sequences are typically less than 25%-40%.

"Sequence identity" is defined as a percentage (e.g., 60%, 65%, 70%, 75%, or 99%), which indicates the percentage of bases that are shared between the compared sequences. This percentage of identity or homology can be determined using specialized software, such as BLAST, particularly BLASTN and BLASTP. Details of these software can be found on the NCBI website.

Sequencing analysis of target nucleic acid sequences can be achieved using a variety of sequencing methods, including but not limited to sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescence in situ sequencing (FISSEQ), FISSEQ beads (U.S. Patent 7,425,431), wobble sequencing (PCT/US05/27695), multiplex sequencing (U.S. Serial No. 12/027,039 filed February 6, 2008; Porreca et al (2007) Nat. Methods 4:931), polyclonal (POLONY) sequencing (U.S. Patent Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425); nanogrid rolling circle sequencing (FISSEQ). Sequencing) (ROLONY) (U.S. Serial No. 12/120,241, filed May 14, 2008), allele-specific oligomer ligation assays (e.g., oligomer ligation assay (OLA), single template molecule OLA using ligated linear probes and rolling circle amplification (RCA) readout, ligated padlock probes, and/or single template molecule OLA using ligated circular padlock probes and rolling circle amplification (RCA) readout), etc. High-throughput sequencing methods can also be used on circular array sequencing, for example, using various platforms (e.g., Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platforms, etc.). High-throughput sequencing methods are described in U.S. Serial No. 61/162,913, filed March 24, 2009. Various light-based sequencing technologies are known in the art (Landegren et al. (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1:95-100; and Shi (2001) Clin. Chem. 47:164-172).

The amplified DNA can be sequenced using a variety of methods. High-throughput sequencing, such as Applied Biosystems' SOLID sequencing method or Illumina's Genome Analyzer sequencing platform, can yield anywhere from 10,000 to 1 billion reads. A "read" in this context refers to a continuous nucleic acid sequence in the sequencing reaction.

Shotgun sequencing is suitable for sequencing large DNA sequences (such as whole genomes). This method first fragments the DNA into individually sequenceable fragments. Sequencing results from these fragments are then reassembled according to the original sequence to form a complete sequence. There are many methods for fragmenting DNA, including enzymatic digestion and mechanical shearing. Overlapping sequences can be compared and analyzed using appropriate bioinformatics software.

Amplification and sequencing methods are useful in predictive medicine, including diagnostic assays, prognostic assays, and pharmacogenomics. Monitoring clinical trials can aid in prognostic (predictive) purposes, thereby enabling preventive treatment of individuals. Accordingly, one aspect of the present invention relates to a diagnostic assay for detecting genomic DNA to determine whether an individual is at risk for developing a condition and/or disease. Such assays can be used for prognostic and/or predictive purposes, thereby enabling preventive treatment of individuals before the onset of a condition and/or disease.

The inventive method also provides a method for identifying the haploid structure of homologous chromosomes. Haploid information plays an important role in describing and interpreting genomic and genetic diversity. Individual haploid information facilitates the use of personalized medicine. Complete individual haploid information also facilitates genome-wide association studies (GWAS) of complex traits. In this regard, genetic material derived from a single cell from an individual, or multiple cells of the same cell type, is separated into multiple fractions or reaction mixtures, so that DNA from homologous chromosomes from the same individual can be separated. Using the amplification, sequencing, and analysis methods described above, each amplified fraction is analyzed for single nucleotide polymorphisms (SNPs) using a genomic sequence, such as the human genome (for details, see International Human Genome Sequencing Consortium, Nature 431, 931-945 (2004)). This method is also applicable to other species with reference genomes. SNP analysis can be used to identify haploids, as described in H.C. Fan et al., Nat. Biotech. 29, 51-57 (2011). Based on the sequence information and single nucleotide polymorphisms, a haplotype of a single cell is constructed, for example, with a size greater than 100 KB. Alternatively, multiple single cell haplotypes from the same individual are compared to determine the complete haplotype of the individual.

The method for determining the haploid type of a single cell involves extracting DNA from tissue material, collecting cells, and collecting single cells and DNA molecules without obvious damage or degradation. The extracted DNA is then divided into multiple fractions and added to the reaction solution, ensuring that only one genomic subfraction is present in each reaction. DNA from homologous chromosomes is then amplified, sequenced, and analyzed for polymorphisms. This is followed by cellular haploid analysis and SNP analysis. Separately, the haploid karyotype of the single-cell genomic DNA is determined by comparing the DNA amplified from each fraction.

When there is no reference sequence for the sample species or some cancer samples have complex structural variations, de novo gene assembly methods (de-novo) are required. De novo genome assembly is accomplished by splicing and mapping the sequencing results obtained from each genome sub-fraction. The de novo genome assembly principle can also be applied to DNA currently extracted from single cells or multiple cells. For example, multiple cells from the same cell type can be separated and DNA extracted, and then the sequencing results can be amplified, sequenced, compared and analyzed separately, and spliced and assembled without a reference sequence to obtain complete genome information.

The term "biological sample" mentioned in the method of this patent includes but is not limited to the following: tissues, cells, biological fluids and isolates obtained from these samples.

In this patented method, "electronic device-readable medium" refers to any electronic device capable of reading and directly identifying any device that stores, maintains, or contains data or information. These media include, but are not limited to, the following: storage disks, such as floppy disks; hard disks; magnetic tapes; optical storage, such as CDs; electronic storage, such as RAM, ROM, EPROM, EEPROM, and the like; conventional hard disks; and combinations of these media, such as magnetic disk/CD storage. These media are used to record one or more expression profiles.

As used herein, the term "electronic device" refers to any suitable computer or device or other data and information storage device that meets the requirements. Examples include: stand-alone computing systems; networks, including local area networks (LANs), wide area networks (WANs), intranets, and extranets; electronic devices such as personal digital assistant systems, cell phones, pagers, etc.; and distributed processing systems.

As used herein, "recording" refers to the process of recording and encoding information on a display screen of an electronic device. A skilled person can easily use existing methods to record information on a device and aggregate the information into a result comprising one or more expression profiles.

The genomic DNA information of the present invention can be recorded in electronic devices using a variety of software systems and programs. For example, nucleotide sequences can be represented as Word files and encoded using commercial software such as WordPerfect and Microsoft Word, or as ASCII files and stored in databases such as DB2, Sybase, Oracle, etc. Various data processing formats (e.g., files or databases) can be used to obtain or create a medium containing one or more expression profiles as described above.

It should be understood that the specific method of the invention described above is merely an illustration of a portion of the principle. Numerous modifications may be made to the method described herein without departing from the essence and scope of the invention. All references, patents, and published patent applications cited in this patent specification are incorporated herein by reference.

The following examples illustrate the invention method. These examples should not be interpreted as limiting the scope of the invention method, because based on the existing disclosure, drawings and related documents, there are other similar methods.

Example 1

Single-cell DNA amplification

Dissociate cells using a laser dissecting microscope .

Select one cell from the cell culture dish, separate the cells using a laser dissecting microscope (LDM-6500, Leica), and suspend them in a centrifuge tube. The separated cells are then transferred to a culture dish for culture, and the cells are observed using a bright field microscope under a 10x objective lens. The membrane surrounding the selected single cell is then removed using an ultraviolet laser and placed in a PCR tube. Centrifuge briefly to allow the cells to settle to the bottom of the tube. Add 5μl of lysis buffer (30mM Tris-Cl PH7.8, 2mM KCl, 0.2% Trition X-100, 12.5μg/ml Qiagen Protease) to one side of the PCR tube and flow into the tube. The captured cells are then lysed in a PCR instrument using the following temperature conditions: 50°C for 3 hours, 75°C for 20 minutes, and 80°C for 5 minutes.

Linear preamplification

In linear preamplification, a pair of quasi-degenerate primers is used to amplify the entire genomic DNA. The primers are NG and NT:

NG5’-GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG-3’

NT5’-GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT-3’

The following solution was added to a PCR tube for the first amplification: 1.5 μl Th _x rmoPol Buffer (NEB), 1.5 μl Φ29 Reaction Buffer (NEB), 1.0 μl dNTP (10 mM), 26 μl H ₂ O (Ambion), 0.1 μl NG and NT primers (50 μM).

After adding PCR buffer to the PCR tube containing the lysed single cells, heat at 94°C for 3 minutes to denature the DNA into single-stranded DNA. Quickly place the sample on ice to allow the temperature to reach 0°C, during which time the primers bind to the template. Add 0.6 μl of a mixture of Bst Large Fragment Polymerase and Φ29 (NEB) to the PCR tube. Run the PCR instrument at the following temperatures and times:

10℃-45 seconds

20℃-45 seconds

30℃-60 seconds

40℃-45 seconds

50℃-45 seconds

62℃-3 minutes

95℃-20 seconds

The PCR tubes were then placed on ice to stop the reaction and start the reaction using new primers.

In the second and subsequent cycles, fresh polymerase mix is added to the PCR tubes and the following cycles are run in the PCR instrument:

10℃-45 seconds

20℃-45 seconds

30℃-60 seconds

40℃-45 seconds

50℃-45 seconds

62℃-3 minutes

95℃-20 seconds

58℃ - at least 20 seconds

Example II

The amplicon from Example 1 was further amplified using standard methods.

Use standard PCR amplification methods to exponentially amplify the product of Example 1. Working on ice, add the following reaction system to a PCR tube:

3.0 μl ThermoPol Buffer (NEB)

1.0 μl dNTP (10 mM),

26 μl H ₂ O (Ambion)

0.1 μl primer (100 μM) (5’-GTGAGTGATGGTTGAGGTAGTGTGGAG-3’)

1.0 μl Deep VentR (exo-) (NEB)

1-2 μg of DNA product was amplified using the following standard PCR procedure.

94℃-20 seconds

59℃-20 seconds

72℃-3 minutes

17 cycles

72℃-5 minutes

4℃-∞

After exponential amplification, the DNA was purified using Qiagen columns and stored, and the primer ends of the DNA amplicons were removed.

Other amplification methods known below may also be used.

One of the most common amplification methods is PCR, which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159, which are incorporated herein by reference. PCR is considered an acceptable method for DNA amplification, and it is important to note that the inventive method may also be performed using other amplification techniques. These techniques are generally considered conventional techniques and are briefly discussed below. In PCR, the process of selective binding of primer pairs to nucleotides occurs under conditions that allow selective binding. In template-dependent amplification, the primers contain nucleotides that can initiate initial nucleotide synthesis. Primers can be in single-stranded or double-stranded form, but single-stranded primers are preferred. In a series of template-dependent amplifications, primers are used to amplify the target gene sequence of a known template.

The target products of the currently disclosed DNA amplification methods are all nucleic acids, which can be any nucleic acid or nucleic acid analog that can be amplified using existing amplification techniques. For example, the target nucleic acids in the context of the inventive method include, but are not limited to, genomic DNA, cDNA, RNA, mRNA, cosmid DNA, BAC DNA, PAC DNA, YAC DNA, and synthetic DNA. Genomic DNA derived from prokaryotic cells, eukaryotic cells, or tissues can be used as a template for the currently disclosed DNA amplification methods. Isolated mRNA with a Poly A tail can be reverse transcribed (RT) into cDNA and then used as a template for DNA amplification. cDNA can be directly used as a DNA template for amplification. In addition, when the starting material is an RNA sample rather than a DNA sample, RNA or mRNA can be directly amplified.

In a PCR reaction, the primer pairs have sequences that complement those of the target gene. If the target gene is present in the template, the primers will bind to it. DNA template:primer complex. Sufficient dNTPs and a DNA polymerase (such as Taq polymerase) are added to the reaction system.

DNA template: After the primer-primer complex is formed, DNA polymerase begins catalyzing the extension of the primer along the target gene sequence. By controlling the temperature of the reaction system, the extended primer is released from the template, forming the reaction product. The remaining primer binds again to the template and the newly formed product, and the previous steps are repeated. This series of steps is called a "cycle." This cycle continues until a sufficient amount of amplicon is produced.

Next, the amplicon needs to be detected. Reliable methods generally use visualization to detect the amplicon. Alternatively, indirect detection can be achieved through fluorescent labeling, chemifluorescent labeling, isotope tracer labeling or labeled nucleotides, mass tags, or electrical or thermal pulse signals.

Another amplification method is the ligase chain reaction (LCR). This method is disclosed in European Patent Application No. 320,308. In the LCR reaction, when exposed to the target gene sequence, two pairs of complementary probes bind to the two template strands, and the two binding partners are adjacent to each other. After the addition of ligase, the two adjacent probes are connected to each other. As with PCR, through temperature cycling, the generated units dissociate from the template and act as new templates to bind to the excess probes. U.S. Patent No. 4,883,750 also includes an amplification method similar to LCR that utilizes the binding of probes to template strands.

The Qbeta replicase method, described in PCT Patent Application No. PCT/US87/00880, is another amplification method. This method involves adding a repetitive RNA sequence complementary to the target gene sequence to a system containing RNA polymerase and a template. RNA polymerase amplifies this repetitive sequence, allowing for detection.

Isothermal amplification uses restriction endonucleases and ligases to amplify target molecules containing 5′-[α-thio]triphosphate sites. This method can also be used for DNA amplification, for example, as described by Walker et al.

Another isothermal amplification method is strand displacement amplification (SDA), which involves multiple strand displacement and synthesis processes. SDA technology is described in U.S. Patents Nos. 5,712, 1245, 648, 211, and 5,455,166. Another similar method is the repair chain reaction (RCR). This method involves mixing multiple primer probes with the target region during annealing, followed by strand repair, which retains two of the four bases. The other two bases are added to the reaction system as biotinylated derivatives. Similar detection methods can also be used in SDA reactions.

A cyclic probe reaction (CPR) can also be used to detect specific target genes. In a CPR reaction, the probe has nonspecific DNA at its 3' and 5' ends, with a specific RNA sequence in between. After the probe binds to the template DNA, RNase H catalyzes the reaction, which digests it to produce a reaction product, also known as the identification product. The initial template anneals and binds to additional probes, initiating a new round of reaction.

Other currently available nucleic acid amplification methods include transcription-based amplification (rAS), nucleic acid sequence-based amplification (NASBA), and 3SR (Kwoh et al., Proc Natl Acad Sci USA, 86:1173-77, 1989; PCT WO88/10315 et al., 1989). In the NASBA method, nucleic acids are obtained through standard phenol-chloroform extraction, heat denaturation, and lysis buffer treatment. DNA and RNA are then separated using minispin columns or RNA is extracted using guanidine hydrochloride. These amplification techniques generally involve the following steps: annealing to allow primers to bind to the template strand, polymerization reaction, RNase H digestion of the DNA/RNA binding product, and high-temperature denaturation of the DNA double strands. Regardless of the technique, single-stranded DNA is converted to double-stranded DNA by adding a primer targeting the second strand to the reaction system for polymerization. The double-stranded DNA can then be transcribed in large quantities using polymerases such as T7 or SP6. In an isothermal cycling reaction, RNA is reverse transcribed into double-stranded DNA, which is then transcribed using polymerase T7 or SP6. The resulting products, whether intact or shortened, are specific for the target gene.

Amplification techniques described in UK Patent No. GB2,202,328 and PCT No. PCT/US89/01025 can also be used for amplification. The former uses modified primers in a PCR-like format and enzyme-dependent synthesis. These primers incorporate capture groups (such as biotin) or detection groups (such as enzymes). The latter involves adding an excess of labeled probes to the reaction system. In the presence of the target sequence, the probes bind to it and undergo catalytic cleavage. After cleavage, the template strand remains intact and can bind to other probes. The signal emitted by the probe cleavage indicates the presence of the target sequence.

Davey et al., European Patent No. 329,822, discloses a method for cyclic amplification of single-stranded RNA (ssRNA), ssDNA, and double-stranded DNA (dsDNA). An oligonucleotide primer first binds to the initial template, ssRNA, and is extended under the catalysis of reverse transcriptase. RNase H then removes the RNA from the DNA:RNA complex. The remaining ssDNA serves as a secondary template, which binds to a second primer containing an RNA polymerase promoter sequence homologous to the 5' end of the template. DNA polymerase then extends the ssDNA to form a double-stranded DNA molecule. This double-stranded DNA contains the same sequence as the original RNA between the primers and also contains a promoter sequence at one end. Under the action of an appropriate RNA polymerase, this promoter sequence can be used to generate numerous RNA copies derived from the DNA. These copies then undergo accelerated amplification after entering the cycle. Therefore, this method can use either DNA or RNA as the starting template, enabling isothermal amplification by selecting the appropriate enzyme, without the need to add fresh enzyme for each cycle.

Miller et al., PCT WO89/06700 discloses an amplification method that uses a promoter/primer sequence to hybridize with a single-stranded DNA of interest to generate multiple products of reverse-transcribed RNA sequences. This method is not cyclic, so no new template is generated during the RNA transcription reaction.

Other amplification methods include race and one-sided PCR (Frohman, In: PCR Protocols: A Guide To Methods And Applications, Academic Press, N.Y., 1990). This method involves ligating two (or more) oligonucleotides containing the desired oligonucleotide sequence and then amplifying the oligonucleotides. Similarly, this method can also be used to amplify DNA (Wu et al., Genomics 4:560-569, 1989).

Example III

Removal of primer sequences from PCR amplification

Using phosphothioate nucleotides is a method for removing primer sequences from the amplicon. The DNA product is then re-amplified using dA, dC, dT, and phosphothioate dG. Because the primers do not contain dC nucleotides, their complementary sequences should not contain dG nucleotides. Nuclease exonuclease III is used to digest the PCR product from the 3' end. However, phosphothioate-containing nucleotides cannot be digested, so the sequence is digested to the phosphothioate-containing dG. Mung Bean exonuclease is also included in the enzyme mixture to eliminate the 5' ends generated during the exonuclease III digestion.

The reaction buffer and reaction conditions are as follows: 1X Buffer 1 (New England Biolabs), 5 U ExoIII, and 0.5 U Mung Bean exonuclease (New England Biolabs). The reaction is incubated at room temperature for 2 minutes. The reaction can then be terminated by adding 5 μl of 0.5 M EDTA.

The following flowchart shows the reaction steps for removing primer sequences from DNA products. After removing the primer sequences, the products can be used for high-throughput sequencing. For example, the primer sequence can be removed from the 3' end and then from the 5' end.

Example IV

Removal of priming sequences from PCR amplicons

Hemimethylated primers are used to facilitate excision of the priming sequence of the amplicon product. Hemimethylated primers are used in the second round of amplification to activate the activity of the MspJI restriction endonuclease or other enzymes with similar properties that require a methylase to recognize the restriction site. The sequences of the hemimethylated primers are as follows:

First round amplification primers:

GT GAG TGA TGG TTG AGG TCT TGT GGA GNNNNNGGG

GT GAG TGA TGG TTG AGG TCT TGT GGA GNNNNNTTT

Second round amplification primers:

GT GAG TGATGG TTGAGG TmCT TGT GGA G

After the second round of amplification, enzyme MspJI (0.5 U), buffer 4 (New England Biolabs), and BSA were added to the purified DNA product. The reaction was incubated at 37°C for 4 hours and then terminated by column purification.

Example V

Useful tags

Including a recognizable group in primers or amplicon, or adding a recognizable group to a probe, facilitates the final qualitative and quantitative analysis of the amplicon. Many different types of labels are available for this purpose, including fluorescent groups, chromophores, radioisotopes, enzyme labels, immunolabeled antibodies, chemiluminescent groups, electroluminescent groups, affinity tags, and many others. Those skilled in the art will be familiar with these and other fluorescent substances not mentioned here.

Examples of affinity tags include, but are not limited to, antibodies, antibody fragments, receptor proteins, hormones, biotin, DNP, or any polypeptide/protein molecule bound to an affinity tag that can be used to isolate a proliferation gene.

Examples of enzyme labels include enzymes such as urease, alkaline phosphatase, or peroxidase. Additionally, colorimetric indicator substrates, visible to the human eye, or spectrophotometric methods can be used to detect specific hybridization with complementary nucleic acid samples. These examples are well known to those skilled in the art and are not limited to the examples listed above.

Among the currently known fluorescent substances, the following are more commonly used: Alexa350, Alexa430, AMCA, BODIPY630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5, 6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green488, OregonGreen500, Oregon Green514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, and Texas Red.

Example VI

Separation technology

After amplification, products of different fragment sizes need to be separated from the amplicon and analyzed to determine whether specific amplification has occurred from the amplicon, the starting template, or excess primers.

In standard laboratory procedures, the amplicons can be separated by electrophoresis using agarose gel, agarose-acrylamide, or polyacrylamide gel (Sambrook et al., "Molecular Cloning," A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, New York, 13.7-13.9: 1989). Gel electrophoresis separation techniques are well known.

Alternatively, separation can be achieved using chromatography. A wide variety of chromatographic methods are available, including adsorption, isolation, ion exchange, and molecular sieves, as well as specialized techniques such as columns, membranes, thin-layer chromatography, and gas chromatography (Freifelder, Physical Biochemstry Applications to Biochemistry and Molecular Biology, 2nd ed. Wm. Freeman and Co., New York, NY, 1982). Another alternative approach involves capturing a tag attached to the nucleic acid product, such as using magnetic beads containing avidin or antibodies to capture nucleic acids labeled with biotin or antigen, respectively.

Microfluidic chip technology includes separation platforms such as capillary tubes (ACLARA BioSciences Inc.) and microchips (Caliper Technologies Inc.). These microfluidic separation platforms require only nanoliter sample volumes, while other separation technologies require microliter sample volumes. Some genetic analysis protocols utilize microfluidic devices. For example, published PCT application No. WO94/05414 (Northrup and White) describes the use of a micro-PCR™ device to isolate and amplify nucleic acids from specimens. U.S. Patents Nos. 5,304,487, 5,296,375, and 5,856,174 describe similar devices and methods.

In some instances, it may be necessary to provide an additional or alternative method for analyzing DNA amplicons. In these instances, microtube arrays are considered for sample analysis. Microtube array electrophoresis typically uses thin capillaries or channels that contain a specific separation medium. Electrophoresis of the sample through the capillaries separates the bands contained in the sample. Microtube array electrophoresis typically provides a method for rapid sample analysis, PCR.TM, product analysis, and enzyme fragment size determination. The high surface-to-volume ratio of these capillaries allows for faster sample separation due to the fact that the higher electric fields passing through the capillaries do not undergo substantial thermal changes. In addition, combined with confocal imaging, this method can increase the sensitivity of the analysis, which is comparable to radioactive sequencing methods. Precision-fabricated microfluidic devices include microcapillary array electrophoresis, which has been discussed in detail above, for example, in Jacobson et al., Anal Chem, 66:1107-1113, 1994; Effenhauser et al., Anal Chem, 66:2949-2953, 1994; Harrison et al., Science, 261:895-897, 1993; Effenhauser et al., Anal Chem, 65:2637-2642, 1993; Manz et al., J. Chromatogr, 593:253-258, 1992; and U.S. Pat. No. 5,904,824, incorporated herein by reference. Typically, these methods involve photolithography of micrometer-scale channels in silica, silica gel, or other crystalline substrates or chips, which can be readily adapted for use with the present invention.

Tsuda et al. (Anal Chem, 62:2149-2152, 1990) introduced rectangular capillaries as an alternative to cylindrical glass capillaries. Advantages of these systems include efficient heat dissipation rates that depend on the ratio of height to width, thus requiring high surface-to-volume ratios and detection modes with highly sensitive optical inlets. These planar separation channels can simultaneously perform two-dimensional separations, one in the separation channel and one in the sample area, enabling detection using multichannel array detectors.

In many capillary electrophoresis methods, a capillary (e.g., fused silica capillary, channel-etched, machined, or formed into a flat substrate) is filled with an appropriate separation region/filtering medium. Generally, a variety of known filter media may be used for microtube array analysis. Such filter media include, for example, hydroxyethyl cellulose, polyacrylamide, agarose, and the like. Typically, microtube array analysis can utilize a variety of filter media, such as hydroxyethyl cellulose, polyacrylamide, dextran, and the like. Specific gel matrices, buffers, and running conditions are selected to maximize separation. For example, the fragment size of the nucleic acid, the desired resolution, and the starting or undenatured nucleic acid molecules may be considered. For example, the running buffer may contain denaturants, high concentrations of agents such as urea to denature the nucleic acid sample, and the like.

Mass spectrometry provides a method for "quantifying" individual molecules by ionizing and evaporating the molecules in a vacuum, allowing them to "fly". Under the combination of electromagnetic fields, the trajectory of the ions depends on their respective mass (m) and charge (z). For low molecular weight molecules, mass spectrometry has become a conventional physical-organic technical means for analyzing and identifying organic molecules and determining the mass of the original molecular ions. In addition, the collision of the original molecular ions with other particles causes the molecular ions to disperse into secondary ions by the so-called collision-induced dissociation (CID) mechanism. Detailed structural information of the original molecular ions can be deduced from this fragmentation pattern/path. Other applications of mass spectrometry technology are summarized in enzymology methods. See Vol. 193: "Mass spectrometry" (j.a.Mccloskey, editor), 1990, Academic press, New York.

Mass spectrometry offers significant advantages for the structural analysis of nucleic acids, as it provides highly sensitive detection, accurate mass measurement, and the ability to derive detailed structural information through CID coupled with an MS/MS configuration and speed, as well as online data transfer directly to a computer. Reviews in this field include (Schram, Methods Biochem Anal, 34:203-287, 1990) and (Crain, Mass Spectrometry Reviews, 9:505-554, 1990). The greatest obstacle to the application of mass spectrometry to nucleic acid analysis is the difficulty in volatilizing these biopolymers. Consequently, "sequencing" has been limited to low-molecular-weight synthetic oligonucleotides, where the mass of the primary molecular ion is determined by mass spectrometry and the sequence is confirmed. Alternatively, to confirm the sequence, the CID MS/MS configuration utilizes secondary ion formation (ion fragmentation), specifically, ionization and volatilization, fast atom bombardment mass spectrometry (Fab mass spectrometry) or plasma desorption mass spectrometry (Pd mass spectrometry). For example, Fab mass spectrometry was used to analyze dimers of chemically synthesized protected oligodeoxynucleotides (Koster et al., Biomedical Environmental Mass Spectrometry 14: 111-116, 1987).

Two ionization/desorption techniques are electrospray/ion spray (ES) and matrix-assisted laser desorption/ionization (MALDI). ES mass spectrometry was introduced by Fenn et al., J. Phys. Chem. 88:4451-59, 1984; PCT Application No. Wo90/14148; for a summary of its applications, see Smith et al., Anal Chem 62:882-89, 1990, and Ardrey, Electrospray mass spectrometry, Spectroscopy Europe, 4:10-18, 1992. As a mass analyzer, the quadrupole is the most commonly used. The accuracy of molecular weight determination for femtogram-level samples depends on the presence of multiple ion peaks that can be used for mass calculations.

In contrast, MALDI mass spectrometry is particularly attractive because time-of-flight (TOF) devices are used as mass analyzers. Hillenkamp et al. introduced MALDI-TOF mass spectrometry (Hillenkamp et al., Biological Mass Spectrometry, eds. Burlingame and McCloskey, Elsevier Science Publishers, Amsterdam, pp. 49-60, 1990). Because this technique does not produce multiple molecular ion peaks in most cases, this type of mass spectrometry is generally considered simpler than ES mass spectrometry. DNA can be desorbed and volatilized at molecular weights up to 410,000 Daltons (Williams et al., Science, 246:1585-87, 1989). Recently, infrared lasers (IR) using this technique have been applied to the analysis of larger nucleic acids, such as synthetic DNA, restriction enzyme fragments of plasmids, and transcribed RNAs up to 2180 nucleotides. (Berkenkamp et al., Science, 281:260-2, 1998) Berkenkamp also described how to use MALDI-TOF IR to analyze DNA and RNA samples from limited purified samples.

Japanese Patent No. 59-131909 describes an instrument that can detect and separate nucleic acid fragments by electrophoresis, liquid chromatography, or high-speed gel filtration. Mass spectrometry detection is achieved by incorporating ions normally absent from DNA, such as S, Br, I, Ag, Au, Pt, Os, and Hg, into nucleic acids.

Fluorescently labeling hybridization oligonucleotide probes is a well-known experimental technique and a sensitive, non-radioactive method for detecting probe hybridization. A more recently established detection method utilizes fluorescence energy transfer (FET) rather than direct fluorescence intensity. FET occurs between a donor fluorophore and an acceptor dye (which may or may not be a fluorophore) when the absorption spectrum of one (the acceptor) overlaps the emission spectrum of the other (the donor), bringing the two dyes into close proximity. Dyes with these properties are referred to as donor/acceptor dye pairs or energy transfer dye pairs. Energy from the donor dye excitation is transferred to the adjacent acceptor via a resonant dipole-induced dipole interaction. This results in quenching of the donor fluorescence. In some cases, if the acceptor is also a fluorophore, their fluorescence intensity is enhanced. The efficiency of energy transfer is largely dependent on the distance between the donor and acceptor, and equations relating these relationships were developed by Forster, Ann Phys 2:55-75, 1948. The distance between the donor and acceptor dyes at which energy transfer efficiency is 50% is defined as the Forster distance (Ro). Other known fluorescence quenching mechanisms include charge transfer and collisional quenching.

Mechanisms such as energy transfer rely on the quenching effect of two dyes interacting in close proximity. This is an effective method for detecting or identifying nucleotide sequences because it allows analysis in a homogeneous reaction system. Homogeneous analysis systems differ from conventional probe hybridization assays, which rely on detecting fluorescent labels derived from a single fluorophore, as diverse assays require the additional step of separating hybridized tags from free tags. Several hybridization analysis modes for FET are described in Nonisotopic DNA Probe Technology (Academic Press, Inc., pp. 311-352, 1992).

A homogeneous assay, utilizing energy transfer or other mechanisms of fluorescence quenching to detect nucleic acid amplification, has been described by Higuchi et al. (Biotechnology 10:413-417, 1992). This method involves real-time detection of DNA amplification by monitoring the fluorescence intensity of ethidium bromide bound to double-stranded DNA. This method has limited sensitivity because ethidium bromide binding is not targeted and background amplification is also detected. Lee et al. (Nucleic Acids Res 21:3761-3766, 1993) disclose a real-time detection method in which a dual-labeled probe is cleaved during PCR amplification of the target gene. The probe hybridizes to the downstream amplification primer, whereupon the probe is hydrolyzed by the 5′-3′ exonuclease of the Taq enzyme, separating the two fluorescent dyes and forming an energy transfer pair. Probe cleavage results in an increase in fluorescence intensity. PCT Publication WO 96/21144 reports a continuous fluorescence assay in which enzymatic nucleic acid cleavage results in fluorescence enhancement. Fluorescence energy transfer (FET) is used when only a single fluorescent tag is present, i.e. by hybridizing the target gene and quenching the fluorescent group.

In nucleic acid amplification detection (U.S. Patent No. 5,547,861), it is described that a primer or probe hybridizes to the downstream of the target gene to form a hybridized amplification primer. The primer is extended similarly to the amplification primer under the action of the polymerase. The extended amplicon replaces the primer in the amplification of the target gene to form a double-stranded secondary amplicon, which can be used as a detection indicator for target amplification. The secondary amplicon can be detected through various tags or reporter groups contained in the primer. The restriction sites in the primer can produce fragmented DNA of a specific size, capture groups, special structures such as triple helices, and recognition sites for double-stranded DNA binding proteins.

Many well-known donor/acceptor dye pairs can be used in the methods of the present invention. These include, but are not limited to, fluorescein isothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TALIC), FITC/texas red.tm. molecular probes, FITC/n-hydroxysuccmimidyl1-pyrenebutyrate (PYB), FITC/eosin isothiocyanate (EITC), n-hydroxysuccinimidyl1-pyrenesulfonate (PYS)/FITC, FITC/rhodamine X, FITC/tetramethylrhodamine (TAMRA), and others. The selection of a specific donor/acceptor fluorescent dye pair is not critical. Energy transfer fluorescence quenching occurs only when the emission wavelength of the donor fluorophore overlaps with the excitation wavelength of the acceptor fluorophore. That is, there must be sufficient spectral overlap between the two dyes for energy transfer, charge transfer, or fluorescence quenching to occur. p-(dimethyl aminophenylazo)benzoic acid (DABCYL) is a non-fluorescent acceptor dye that effectively quenches the fluorescence of adjacent fluorophores, such as fluorescein or 5-(2′-aminoethyl)aminonaphthalene (edans). Any dye pair that can produce fluorescence quenching in nucleic acid detection methods is suitable for use in this inventive method. Known endpoint and internal marker detection methods can be used in this inventive method by linking the fluorescence signals of the nucleic acid donor and acceptor dyes.

It is noteworthy that this technology can be used in microarray and/or chip-based DNA technology, as described in the following references: Hacia et al., Nature Genet, 14:441-449, 1996 and Shoemaker et al., Nature Genetics, 14:450-456, 1996. These methods include accurate and rapid quantitative analysis of multiple genes. By labeling genes with oligonucleotides or using fixed probe arrays, chip technology can be used to sort target molecules by high-throughput hybridization (Pease et al., Proc Natl Acad Sci USA, 91:5022-5026, 1994; Fodor et al., Nature, 364:555-556, 1993).

Furthermore, products amplified using this technology can be used in Bistar's OIA technology. OIA utilizes a mirror-like silicon substrate. A thin film optical coating and capture antibodies are adsorbed onto this silicon substrate. White light shines on the film, creating a golden background color. This color persists until the thickness of the optical film is altered.

When a positive sample is placed on this silica substrate, the ligand and antibody bind. As the ligand is added to achieve mass enhancement, the color changes from gold to purple/blue due to the increased thickness of the molecular layer. This technology can be found in U.S. Patent No. 5,541,057, which is incorporated herein by reference.

Added RNA or DNA can be quantified using real-time quantitative PCR (RT-PCR) (Higuchi et al., Biotechnology 10:413-417, 1992). By determining the concentration of amplicon after a certain number of amplification cycles and within the linear region, the relative concentration of the target sequence in the original mixture can be determined. For example, if the DNA mixture is cDNA transcribed from RNA extracted from tissues or cells, the relative abundance of target mRNA containing the target sequence can be determined in this way. The relative proportionality of the target fragment concentration only holds true within the linear region of the amplification reaction.

The final amplicon concentration of the target DNA during the plateau phase of the amplification curve is determined by the adequacy of the reaction reagents and is independent of the initial target DNA concentration. Therefore, the first requirement for accurate relative quantification of RNA or DNA by real-time quantitative PCR is that the concentration of the amplicon be within the linear region of the reaction. A second requirement is that the cDNA of the specific mRNA sequence amplified during the RT-PCR reaction be normalized to an independent standard sample. The goal of an RT-PCR experiment is to determine the abundance of a specific RNA or DNA sequence relative to the total RNA or DNA in the sample.

Luminex technology enables the quantification of nucleic acids immobilized on colored microspheres. The intensity of the biomolecular reaction is measured by measuring a second molecule called a reporter. The reporter molecule is attached to the microsphere to measure the intensity of the reaction. Because both the microsphere and the reporter molecule are colored, digital signal processing can interpret these signals in real time to quantify each reaction in real time. These standardized technologies can be found in U.S. Patent Nos. 5,736,303 and 6,057,107, which are hereby incorporated by reference.

Example VII

Detection technology

The amplicon must be detected to confirm whether the target gene has been amplified. One method for visualization is gel imaging using fluorescent dyes, such as ethidium bromide or Vistra Green, followed by UV illumination. Alternatively, if the amplicon itself is radioactively or fluorescently labeled with nucleotides, the amplified product can be directly exposed to X-ray film or detected using appropriate excitation spectra.

In one embodiment, visualization is achieved indirectly using a nucleic acid probe. After separation of the amplicon, a labeled nucleic acid probe is mixed with the amplicon. It is optimal for the probe to be bound to a dye molecule, but radioactive labeling can also be used. In another embodiment, the probe is bound to a binding molecule, such as an antibody or biotin, or other detectable ligand. In another embodiment, the probe contains a fluorescent dye or label. In another embodiment, the probe contains a mass marker to detect the amplified molecule. Other foreseeable implementations include Taqman™ and molecular beacon probes. In another embodiment, solid-state capture methods combined with standardized probes can also be used.

The type of labeling molecule incorporated into the DNA amplicon depends on the analytical method. When separation is performed using microtube electrophoresis, microfluidics electrophoresis, high-performance liquid chromatography (HPLC), or liquid chromatography (LC), mixed or intercalated fluorescent dyes are used to label and detect the amplicon. The sample is detected dynamically, and the labeled molecule passes through the detector and fluorescence is quantified. If any electrophoresis, HPLC, or LC method is used for separation, the product can be detected by UV absorption, which is an intrinsic property of DNA, so no additional labeling is required. If polyacrylamide gel or slab gel electrophoresis is used, the primers for nucleic acid amplification can be labeled with fluorescent dyes, chromophore dyes, radioactive groups, or detected by enzyme-catalyzed reactions. Enzymatic reactions involve enzyme binding to the primers, for example, after gel separation, analysis by biotin:avidin interaction, or chemical reactions such as luminol chemiluminescence. The fluorescent signal can be monitored dynamically. Radioisotope or non-reactive detection requires gel separation followed by transfer of the DNA molecules to a solid support. If the amplicon is separated by mass spectrometry, labeling is not required, as the nucleic acids can be detected directly.

The various separation methods mentioned above can be combined to apply two or more different properties to a molecule. For example, some PCR primers can be conjugated to ligands for affinity capture, while others may be unlabeled. Labels can include carbohydrates (e.g., for binding to a lectin column), hydrophobic groups (e.g., for binding to a reverse-phase column), biotin (e.g., for binding to a streptavidin column), or antigens (e.g., for binding to an antibody column). The sample is passed through the affinity chromatography column. The flow-through fraction is collected, and the bound fraction is eluted (via chemical cleavage, salt elution, etc.). These samples are further separated by various properties (e.g., mass) to identify the essential components.

Example VIII

Reagent test kit

The material or reagent in the amplification method that shows here can be combined into test kit.Existing amplification test kit that shows roughly comprises at least enzyme and nucleotide and primer sequence that can be reacted.In a preferred embodiment, test kit comprises the specification sheets of DNA amplification from DNA sample.

The test kit relevant to the display method here can generally include one or more preselected primers and/or probe sequences, and these sequences may specifically or non-specifically detect genes to be amplified. Test kit is more likely to comprise one or more nucleic acid probes and/or primer sets to detect nucleic acid. In some implementations, as in the test kit for amplifying nucleic acid, the method for detecting nucleic acid is a mark, such as a fluorescent chromophore, a radioactive label, an enzyme label or the like, these marks and nucleic acid primers or the sequence itself. It is foreseeable that test kit may contain multiple primer combinations to realize the various amplification reactions of this display method. It is also foreseeable that test kit may contain a precipitating reagent so that subsequent DNA samples are preserved with solid media.

Here, a whole genome amplification kit is used as an example. The kit is preferably implemented as follows: a primer with a defined 5' end, a random middle, and a fixed 3' end is provided, and the primer hybridizes uniformly or significantly uniformly with genomic DNA. The kit may include a second primer that has the same fixed sequence as the 5' end of the first primer. For example, the kit can be used to amplify all genes and chromosomal regions, the sequences of which may or may not be known for a particular species. The kit will also include enzymes suitable for amplifying nucleic acids, nucleotides, and the buffer required for amplification.

The test kit in this demonstration method may contain primers modified with one or more complexes. On the other hand, the test kit contains one or more polynucleotide base sequences that do not, or do not significantly, base pair with other sequences in the same reaction. The test kit may contain a DNA polymerase, or a polymerase with strand displacement activity, including, for example, one of the following polymerases, or a combination thereof: Phi29 polymerase, Bst polymerase, Pyrophase 3173 polymerase, Vent polymerase, Deep Vent polymerase, TOPOTaq polymerase, Vent exo-polymerase, DeepVent Exo-polymerase, 9'Nm polymerase, Klenow fragment, MMLV reverse transcriptase, AMV reverse transcriptase, HIV reverse transcriptase, exo-mutants of T7phase DNA polymerase.

The kit may contain separate containers for each reagent and enzyme, as well as probes and primers. Each biological reagent should be dispensed into a separate, appropriate container. These containers typically include at least one test tube or vial. Flasks, reagent bottles, or other reagent containers may also be used. These containers are sealed and stored for commercial distribution. Appropriate larger containers may be used to contain injection aids that match the test tubes. Detailed operating instructions may be provided with the kit.

Example IX

Amplicon sequencing

Using published or well-known methods and processes for high-throughput sequencing, the DNA amplification product can be used for high-throughput sequencing after removing the primer sequence.

Example X

Whole genome amplification and sequencing using a portion of the genetic material of a single cell for genome-wide haplotype detection

A single cell can be isolated and placed in a PCR tube using a mouth pipette or other well-known methods such as laser microdissection and flow cytometry. The PCR tube can then be centrifuged to remove the cells to the bottom of the tube.

A 1-μl blood sample was obtained by fingerstick sampling and placed in 200 μl of red blood cell lysis buffer (0.1% Triton X-100, 10 mM EDTA in PBS). After 5 minutes at room temperature, most red blood cells were lysed, while the nuclei of white blood cells and other cells remained intact. A single cell was then aspirated from the lysis buffer using a mouth pipette and placed in a test tube.

60ul of lysis buffer (30mM Tris-Cl pH 7.8, 2mM EDTA, 20mM KCL, 0.3% Triton X-100, 30mM dTT, 12.5ug/ml Qiagen Protease, 0.1ul of primer 5'-GT GAG TGA TGG TTG AGG TAG TGTGGA GNNNNNGGG-3') is added to the tube containing the single cell. The following temperature cycle is used for cell lysis: 50°C for 3 hours, 70°C for 20 minutes. The lysis buffer and mild heating process are used to reduce double-strand breaks in DNA and the formation of gaps on single strands. The average length of DNA treated in this way is longer than 100kb. The purpose of adding 0.1uM primer to the lysis buffer is to adsorb to the walls of the tube and the pipette to prevent excessive genomic DNA from adsorbing to these surfaces. For tissue samples, the tissue can be ground or a portion of the tissue can be separated by laser microdissection and then placed in 60ul of lysis buffer. For multiple cells, you can first centrifuge to enrich the cells and then lyse them with 60ul of lysis buffer.

The following procedure is used to separate lysed DNA molecules into multiple fractions, each containing a portion of the genome. Single-cell lysate DNA is dispersed in 60 μl of lysis buffer. Various methods can be used to fractionate the lysate into N fractions. As a result, the two alleles are typically separated into distinct fractions. For diploid cells, the chance of the two alleles not being separated into distinct fractions is 1/N. In single-cell haplotype experiments, N can be arbitrarily large to ensure complete allele separation. If multiple identical cells can be collected, the same procedure can be applied to the different cells to ensure allele separation in at least some of the cells. In one aspect, low-adhesion PCR tubes and pipettes are used to prevent DNA from adhering to the tube walls. Amplification primers are added to the lysis buffer to prevent genomic DNA from adhering to the tube walls. After the single-cell lysis step, the separated DNA can be aliquoted into N separate fractions using a pipette. Alternatively, the DNA can be aliquoted into N individual fractions using microfluidics or other known methods. The separated DNA is gently agitated in a microfluidic device at 4°C. The microfluidic device is designed with multiple outlet channels (approximately 30 channels). The solution in each channel is then collected for whole genome amplification. Alternatively, for samples of multiple cells or tissues, the isolated DNA can be diluted to less than the amount of genomic DNA from a single cell, which is approximately 6 pg.

The DNA from each fraction is then subjected to whole genome amplification using the methods described herein. Alternatively, the DNA from each fraction can be amplified using various known methods, such as multiple strand displacement amplification (MDA), or using commercially available whole genome amplification kits such as Picoplex (Rubicon Genomics), Genomeplex (Sigma Aldrich), GenomiPhi (GE Healthcare Lifesciences), or similar products.

Regional haplotype analysis can be performed by separately measuring single-base nucleotide polymorphisms (SNPs) within the genome of each subcellular fraction. Whole-genome haplotype analysis can be achieved by comparing regional haplotypes across multiple fractions. First, the genomes of each subcellular fraction are sequenced and determined. These sequences are aligned to a human reference genome to determine the SNP profile of each subcellular fraction. Because each subcellular fraction contains only a small portion of the single-cell genome, the sequencing coverage of each fraction will be distributed as a block, with large portions of the genome left uncovered. Statistically, all sequencing reads belonging to a block originate from the same chromosome, so all SNPs found within the same block can be linked and assigned to the same haplotype. The length of the haplotype-linked SNPs is determined by the length of the DNA fragments extracted. Whole-genome or whole-chromosome haplotypes can be obtained by repeating the above steps for multiple cells. In this case, the haplotypes obtained for each cell overlap, allowing accurate haplotype assignment for SNPs on a single chromosome. In addition to genome sequencing, other genomic profiling methods (such as SNP microarrays) can also be used.

Demonstrated here is a method for de novo whole-genome assembly by assembling each subcellular haplotype component de novo and comparing them to each other. De novo genome assembly is necessary for species without a reference genome or in situations where genomic variation is significant (e.g., cancer). After sequencing, the sequence of each subcellular component is determined separately. The sequences of these subcellular components can be obtained using conventional de novo assembly programs or algorithms, such as CLCGenomics Workbench (CLC bio), SeqManNGen (DNASTAR), or similar algorithms. The size of the DNA modules is typically around 100 kb. These assembled modules are then aligned to create the assembled genome. Because these modules are of similar size, around 100 kb, the complexity of the genome assembly is proportional to the genome size, while the complexity of traditional genome assembly is exponentially proportional to the genome size.

Example XI

Single-cell haplotype typing

A single white blood cell obtained from de-identified patient P2 was isolated by mouth pipette and lysed in 10 μl of lysis buffer. The tube containing the lysis buffer was placed in a thermal cycler: 50°C for 3 hours, 70°C for 20 minutes. 60 μl of lysis buffer (without enzyme) was added, and the total solution of 70 μl was mixed and divided into 24 PCR tubes. The DNA in each of the 24 PCR tubes was amplified using DeepVentR (exo-) polymerase using the method described in Example 1. After purification, the PCR products were tested for two loci on chromosomes 1 and 2 using fluorescent quantitative PCR. Table 1 shows the test results of the chromosome 1 and chromosome 2 loci in the 24 PCR tubes. The numbers shown in the table are the Ct values from qPCR, indicating a positive result for the locus tested. An 'x' indicates a negative result for the locus tested.

Table 1

tube 1 2 3 4 5 6 7 8 9 10 11 12 chrl x x x x x 18.1 x x x x x x chr2 x x x x x x x x x x x x tube 13 14 15 16 17 18 19 20 twenty one twenty two twenty three twenty four chrl x x x x x x x x x x x 20.8 chr2 twenty two x x x x x x x 20.2 x x x

Both loci showed positive results in both tubes, indicating that allele separation and amplification were successful for all four loci (two pairs of alleles on chromosomes 1 and 2). If a locus is not amplified in an experiment, this could be due to losses during DNA transfer, random amplification, or failure of allele separation. However, the method presented here reduces the likelihood of these events.

Example XII

Haplotype analysis using two cells

Two white blood cells obtained from de-identified patient P2 were separated by mouth pipette and lysed in 10 μl of lysis buffer. The tube containing the lysis buffer was placed in a thermal cycler: 50°C for 3 hours, 70°C for 20 minutes. 60 μl of lysis buffer (without enzyme) was added, and the total solution of 70 μl was mixed and divided into 12 PCR tubes. The DNA in each of the 12 PCR tubes was amplified using DeepVentR (exo-) polymerase using the method described in Example 1. After purification, the PCR products were tested by fluorescent quantitative PCR at eight loci located on chromosomes 1-7 and 9. Table 2 shows the results of staining these loci in the 12 PCR tubes. The numbers shown in the table are the Ct values from the qPCR, indicating a positive result for the locus tested. An 'x' indicates a negative result for the locus tested.

Table 2

tube 1 2 3 4 5 6 7 8 9 10 11 12 chr1 x x x 20.5 x twenty one x x 20.8 20.2 x x chr2 x x x x x x 22.5 x 21.7 26 x 25.2 chr3 x x x x x x x x x x x x chr4 x 22.2 x x twenty three x 24.7 x 22.5 x x x chr5 23.5 x x twenty two x x x x x x x x chr6 25.4 26.4 x x x x x x 23.1 x x x chr7 x x x 23.1 x x 23.4 25.3 x 24.5 x x chr9 25 x x x x 25.5 24.8 x 22.6 x x x

For loci on chromosomes 1, 2, 4, 7, and 9, all four tubes showed positive results, indicating that both pairs of alleles from the two cells were separated in four of the 12 tubes. For chromosome 3, no loci were positive, indicating incomplete amplification at this locus. For loci on chromosomes 5 and 6, fewer than four tubes showed positive results. This may be due to some alleles not being amplified or more than two alleles being separated in the same tube. Increasing the number of tubes can ensure that the majority of alleles are separated in different tubes.

Example XIII

Haplotype typing without isolating single cells

1ul of blood from volunteer P1 was placed in 100ul of cell lysis buffer and the following temperature cycle was set: 50°C for 3 hours, 70°C for 20 minutes. The lysed blood was diluted 400x and 1000x, and 1ul of the diluted solution was used for amplification. After dilution, each tube contained approximately 1/4 and 1/10 of the genomic DNA of a cell. Table 3 shows the results of fluorescence quantification of 1/4 cells (1-5) and 1/10 cells (6-10). The test sites are chromosomes 1-7 and above 9. The numbers in the table below show the Ct values of qPCR, indicating positive results for the sites.

Table 3

The uniform distribution of loci indicates that chromosome entanglement is not a significant factor in allele segregation by dilution. The segregation of alleles on chromosomes 2, 3, 6, 7, and 9 indicates that single-cell segregation is not a requirement for single-cell haplotype typing.

Example XIV

Single-cell whole genome amplification

The Multiple Annealing Loop Amplification Cycle (MALBAC) protocol was performed as follows. SW480 intestinal adenocarcinoma cell line was obtained from the American Type Culture Collection (ATCC, Rockville). SW480 cells were cultured in ATCC-prepared Leibovitz L-15 medium supplemented with 10% fetal bovine serum (ATCC), 100 IU/ml penicillin, and 100 μg/ml streptomycin (ATCC). Cells were treated with 0.25% trypsin-EDTA and then plated on glass slides with PEN membranes (Leica). Cells were fixed with 70% ethanol for three minutes and then rinsed with PBS. Cells were then stained with 0.5% methylene green for approximately 20 seconds and rinsed twice with ddH2O.

Before laser cutting, DNA contamination was removed from low-adhesion PCR tubes using UV irradiation. Single cells were then excised into individual PCR tubes using a laser cutter (Leica LMD7000). After a brief centrifugation to remove the single cells to the bottom of the PCR tubes, 5 μl of freshly prepared cell lysis buffer (30 mM Tris-Cl pH 7.8, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100, 12.5 μg/ml QIAGEN Protease) was added to each tube. Single cells were lysed using the following temperature cycle: 50°C for 3 hours, 75°C for 20 minutes, and 80°C for 5 minutes. Single-cell isolation can also be performed using other methods, such as using a mouth pipette or flow cytometry.

In the pre-amplification, 30 μl of pre-amplification buffer (20 mM Tris-Cl pH 8.8, 10 mM (NH4)2SO4, 10 mM KCl, 3 mM MgSO4, 0.1% Triton X-100, 0.32 μM GAT3T primer (5'-GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNTTT-3'), 0.25uMGAT3G primer (NG5'-GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNGGG-3'), 94C treatment for three minutes to dissociate the double-stranded genomic DNA into single strands. Subsequently, these single-stranded DNAs were quickly placed on ice to ensure their effective binding to the primers. Subsequently, 2.5U of Bst large fragment (NEB), or 2U of Bst large fragment and 0.8U of Pyrophage3173exo- (Lucigen) were added. The following temperature cycle was performed: 10℃-45 seconds, 20℃-45 seconds, 30℃-60 seconds, 40℃-45 seconds, 50℃-45 seconds, 62℃-2 minutes, 95℃-20 seconds, and then the PCR tube was quickly placed on ice.

After annealing on ice, the same polymerase mixture was added and the following temperature cycle was performed: 10°C for 45 seconds, 20°C for 45 seconds, 30°C for 60 seconds, 40°C for 45 seconds, 50°C for 45 seconds, 62°C for 2 minutes, 95°C for 20 seconds, and 58°C for 20 seconds. The above steps were repeated four times to obtain a mixture with the amplified product.

After several cycles of this process, the product may contain either a complete amplicon or a half-amplicon. Annealing at 58°C after melting allows the ends of the amplicon to form double-stranded DNA. A restriction endonuclease (such as BseGI) that recognizes the primer sequence can be used to treat the amplicon. As a result, only the half-amplicon at the half end remains intact. The restriction endonuclease can be inactivated by heating at 80°C for 20 minutes, and then further amplification cycles can be performed to produce amplicons. This method can produce linear amplification results.

The amplified product can be further amplified by PCR for next-generation genome sequencing. To the pre-amplified product, 30 μl of freshly prepared amplification mix (20 mM Tris-Cl pH 8.8, 10 mM (NH₄)₂SO₄, 10 mM KCl, 4 mM MgSO₄, 0.1% Triton X-100, 0.66 μM Bio-GAT primer (5'/Biosg/GTGAGTGATGGTTGAGGTAGTGTGGAG-3'), and 2.4 U Deep VentR exo-polymerase (NEB)) was added. The temperature was cycled 18 times: 94°C for 20 seconds, 59°C for 20 seconds, 65°C for 1 minute, and 72°C for 2 minutes. Starting from one cell, a total of 2-3 μg of double-stranded DNA product was obtained for high-throughput sequencing.

The lengths of the amplicons ranged from 500 bp to 1500 bp, with a 5' biotin modification. Quantitative PCR was performed at 16 loci, each from a different chromosome (Rochette et al., Journal of Molecular Biology 352, 44 (2005)). Amplification was successful at 14 of the 16 sites tested, a success rate consistent with the 92% site coverage achieved by 30X deep whole-genome sequencing. The data are summarized in Table 4 below, showing the Ct values for the 16 randomly selected loci.

Table 4

qPCR Chr1 Chr2 Chr3 Chr4 Chr5 Chr6 Chr7 Chr9 Single cell 22.5 24.4 36.5 24.8 25.4 26.9 25.5 25.2

Positive control 22.2 24.5 29.6 24.4 24.4 24.4 25.5 25.1 qPCR Chr12 Chr13 Chr13 Chr15 Chr16 Chr17 Chr18 Chr19 Single cell 25.8 23.9 39.0 24.7 21.3 24.3 24.2 27.0 Positive control 26.4 26.3 30.0 23.8 20.0 25.8 23.7 22.5

The results of single-cell amplification were consistent with those of the positive sample amplification using 500pg of genomic DNA. The Ct values of all sites in the negative control were greater than 30. The primer information for these sites is as follows:

Chr1+: AGG AAA GGC ATA CTG GAG GGA CAT

Chr1-: TTA GGG ATG GCA CCA CAC TCT TGA

Chr2+:TCC CAG AGA AGC ATC CTC CAT GTT

Chr2-:CAC CAC ACT GCC TCAAAT GTT GCT

Chr3+: TCA AGT TGC CAG CTG TGG CTG TAT

Chr3-:AGA AGG GCA TTT CCT GTC AGT GGA

Chr4+:ATG GGC AAA TCC AGA AGA GTC CAG

Chr4-: CCA TTC ACT TCC TTG GAA AGG TAG CC

Chr5+: AAT AGC GTG CAG TTC TGG GTA GCA

Chr5-：TTC ACA TCC TGG GAG GAA CAG CAT

Chr6+: TGA ATG CCA GGG TGA GAC CTT TGA

Chr6-: TGT TCA TTA TCC CAC GCC AGG ACT

Chr7+: ACC AAA GGA AAG CCA GCC AGT CTA

Chr7-：ACT CCA CAG CTC CCA AGC ATA CAA

Chr9+: TCC CAG CTC TCT CTC TTG CAT CTT

Chr9-:AGT GAA GCT GGT GTATGC AGA GGT

Chr12+: AGA GGG CTG CTT TAT GCA GGT G

Chr12-：CTA CAT TTG GGT CTT TGC TGC CAT G

Chr13+: AGCAGC CCCAGG CAGAT

Chr13-: CGGAGA GGA CGG TCA CGT TTA C

Chr14+: GTC CAG CAC TAG TGA TCT TGT CC

Chr14-: CGT GGG AGT TTT GAAATG CGA TGT

Chr15+: CCT GTC TCT GCT CCT GCG

Chr15-: TGCACA CAT GCA CAG TGGAG

Chr16+: CTC CAA GGT TCT GCA GCC TC

Chr16-：GGTATGACTACA CAT TCA GGC TGG

Chr17+: GTG GTA CAT AGT GCA TGG TCC G

Chr17-：GGC GAC ATA CCC CAA CTT CAT AAG

Chr18+: CGT TCT TAG GAC CAAAGG GCT G

Chr18-:CCA GCA TCC ATG TCT CTG CAC

Chr19+: GCC CAG AGC GCC TGA

Chr19-：CCAG CCC CTG GAC CAC T

The uniformity of amplification can be measured using the Lorentz plot in Figure 5A. Figure 5A shows the relationship between the cumulative number of reads and the cumulative genome coverage, comparing unamplified positive population samples, MALBAC amplified samples based on the present invention, and MDA amplified samples. A completely perfect amplification will form a diagonal line, while a larger deviation from the diagonal line shows that there is a significant bias in amplification. The unamplified positive samples, MALBAC amplification products, and MDA amplification products compared in Figure 5A were all normalized to 8x sequencing depth. The unamplified positive samples are the closest to the diagonal line. At 8x sequencing depth, the MALBAC product coverage reaches 85%, which is similar to 95% of the positive samples and better than 45% of MDA (here, Figure 5A shows that the uncovered portion of the genome is 5%, 15%, and 55% for the positive samples, respectively, for MALBAC and MDA).

CNVs refer to insertions, deletions, and duplications of genomic fragments, ranging in size from a few kilobases to entire chromosomes. CNVs are widespread and found in nearly all tumor types. CNVs originate from a single cell and create genomic variations that are important for tumor development and progression. The method presented here enables single-copy resolution for single-cell CNV analysis. To further demonstrate the uniformity of genomic amplification, we present the power spectrum of read density, which displays the spatial frequency of reads. A perfectly uniform distribution would produce a power spectrum that is a delta function. Analysis shows that the power spectrum of MALBAC-amplified samples is similar to that of unamplified positive samples. MDA, on the other hand, displays high values in the range of tens of kilobases to tens of megabases, indicating both over- and under-amplification of the genome at this scale. This demonstrates the feasibility of the MALBAC method for detecting copy number variations (CNVs).

Due to the uniformity of MALBAC amplification and its single-copy resolution, whole-genome CNVs could be determined between two SW480 cancer cells. These two cells differed from each other, exhibiting digital copy number differences. Without single-cell measurements, this information would be obscured by population measurements. We used an implicit Markov model to identify statistically significant single-cell CNVs. As a control, we obtained the karyotype of the cell line. For example, in diploid cells, most chromosomes have two copies. However, in the SW480 cell line, chromosome 8 has only one copy and chromosome 17 has three copies. The digital counts and karyotype results were consistent.

To further investigate CNVs in single cells, we calculated the relative ratio of whole-genome CNV values between the two cells. This normalization method removes any remaining amplification bias, resulting in smoother CNV results. Approximately eight distinct CNVs were identified genome-wide, indicating eight significant CNV differences between the two cancer cells. This analysis revealed that cell 1 had three copies of chromosome 13, while cell 2 had only two copies.

The amplified DNA products were used to construct libraries for the SoliD and Illumina sequencing systems. After DNA fragmentation, the ends were blunted, and biotinylation at the 5' end prevented efficient ligation of sequencing adapters and amplification adapters. This significantly reduced the proportion of reads that could not be aligned to the reference genome due to amplification primers.

Single-base variants (SNVs) in single cells of the SW480 cell line can be analyzed in this way. While a specific SNV may occur in a single cell, it cannot be detected by sequencing the entire population. This demonstrates the importance of single-cell SNV analysis. Each single cell contained 1.6x10^6 SNVs (Table 5), consistent with a population frequency of approximately one SNV per 1 kb.

Table 5

Most SNVs matched those in the cell population, indicating that these were common SNVs inherited from earlier generations of the cell line. However, amplification results in an error rate of approximately 10^-5, which results in a much higher false positive rate for SNVs than the rate of newly grown SNVs in single cells. This is a significant challenge in single-cell sequencing. To address this issue and clearly identify SNVs in single cells, we sequenced and analyzed two daughter cells derived from a single cell division (Table 1). The chance of finding the same SNV in both daughter cells is approximately 10^-10, which is essentially negligible. We discovered 13 newly generated SNVs. These findings would not have been discovered through genome sequencing of cell populations.

Analysis revealed that 6 of the 13 newly generated SNVs were clustered within a 500-bp region, indicating that their generation was not random. Sequencing data were generated using an Illumina sequencer and aligned using BWA (reference: Bionformatics 25, 1754 (2009); SolidD-generated sequencing data were aligned using BioScope). After alignment, duplicate sequences were removed. SNVs with a phred quality greater than 50 and a site frequency greater than or equal to 0.5 were then detected using vcftools. For each daughter cell pair, 2,459,529 and 2,225,969 SNVs were detected, respectively. Of these, 1,774,887 SNVs were present in both daughter cells. We further screened for SNVs that were present at a sequencing depth of 10x or greater. To identify newly generated SNVs, we screened the SNVs using unamplified population data. SNVs present in both daughter cells but not in the population were potentially newly generated SNVs. However, some false positives still occur due to systematic errors and inaccuracies in amplification and sequencing. These false positive SNVs can be identified and eliminated by amplifying and sequencing an unrelated single-cell sample. After this, the remaining newly generated SNVs need to have at least 20x genome coverage.

The error rate of this amplification method is calculated by comparing SNVs detected in single cells with SNVs in unamplified samples from the population. SNVs detected in single cells but not in the population are mostly due to amplification errors. For a pair of daughter cells, we found 51,805 and 42,705 false positives, respectively, indicating an amplification error rate of 1.7x10^-5 and 1.4x10^-5, respectively.

Single-cell copy number variation was captured using an implicit Markov model. We simulated reads using wgsim to correct for alignment, ensuring an average length of 250 kb and identical reads per window. For each window, we allowed 0–9 copy numbers. We calculated the copy number of cells with deviations from diploidy using a transformation matrix. Specifically, the probability of a transformation matrix from copy number m to copy number n is:

T(m,n)={1-f/b, when m=n=2

1-b/l, when m=n≠2

(b-f)/l, when m≠n=2

f/(N-2)/l, for the rest of the cases

Where f = 10 ^-9 is the frequency of copy number variation, 1 = 5x10 ⁷ is the length of the copy number variation, b = 250000 is the window size, and N = 10 is the number of states.

The probability of transition in the transformation matrix is obtained from a single normal blood cell through linear pre-amplification. It is assumed here that this cell has no significant copy number variation. P2 indicates that the reading in a certain window is diploid. P1 indicates that the corresponding observed reading is haploid. Assume that the diploid read value is composed of an independent combination of haploid read values. Then P2=P1*P1, * represents convolution. This relationship can be reversed in Fourier space. Once p1 is obtained, the transition probability of the high energy level of the transformation matrix can be obtained as Pn=Pn-1*P1. Considering the sequencing depth, the total depth of the reads of cancer cells is normalized by the corresponding depth of normal cells when calculating, and then the most likely state is calculated using the Viterbi algorithm.

Lorentz curves were calculated for the unamplified population, MALBAC, and MDA. Read counts were calculated within 5 kb intervals to generate the Lorentz curves, so amplification bias within 5 kb was averaged out. The portion of the Lorentz curve greater than 5 kb indicates amplification bias above 5 kb. MALBAC's amplification bias is primarily within 5 kb, while the improvement in the 5 kb Lorentz curve for MDA is less pronounced, indicating that MDA has amplification bias much greater than 5 kb.

The results of the sequencing experiments are summarized in Table 6. The SW480 genome is incomplete, encompassing only approximately 90% of the reference genome. Single-cell coverage ratios were normalized using this ratio. *The simultaneous use of Bst large fragments and Pyrophage3173 (exo-) for MALBAC amplification improved coverage compared to amplification using Bst large fragments alone. **S: Data obtained with the SolidD system; I: Data obtained with the Illumina system.

Table 6

Example XV

Prenatal diagnosis

According to certain aspects of the methods disclosed herein, non-invasive prenatal testing can be performed by amplifying genetic material, such as DNA from a fetal cell, a subset of fetal cells, or isolated from the mother's body. According to certain aspects, information about the entire fetal genome, or a significant portion of the genome, can be obtained and analyzed, for example, to identify abnormal or disease-causing genes.

According to certain other aspects, methods for preimplantation genetic screening (PGS) and diagnosis (PGD) are provided herein. More than 100,000 in vitro fertilization (IVF) procedures are performed in the United States each year, and it is very important to be able to screen embryos to be implanted. Current PGS methods include biopsies of polar bodies, blastomeres, blastocysts, etc., followed by various genetic screening methods such as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) to screen for certain specific chromosomal variations (such as trisomy 21), or to detect certain identified genetic mutations with clear phenotypes. In IVF, it is currently known that embryos will pass through a 2 to 12 cell stage before being implanted. According to certain aspects, the genetic material (such as DNA) of one or more cells in the embryo is extracted and amplified so that the full genome of the embryo can be obtained and analyzed, and information on the abnormality and pathogenicity of the embryo can be obtained.

According to certain aspects, nucleated fetal cells can be isolated from maternal blood. Nucleic acids, such as DNA, can be isolated from a single or multiple nucleated fetal cells. Extracellular fetal nucleic acid material can also be obtained from maternal blood. See Lo et al., Nature Reviews Genetics, Vol. 8, pp. 71-77 (2007) and Lo et al., Sci. Transl. Med. 2, 61ra91 (2010), for reference. These nucleic acids can be linearly amplified using the methods described herein. For example, the entire genome of the fetus, or specific sites on the genome, can be amplified. This can be used to identify genomic findings associated with a disease or known phenotype.

Nucleated fetal cells can be obtained by the following methods: Blood is drawn from the mother's vein or by fingerstick sampling. Between 0.1ml and 100ml of maternal blood is drawn. Nucleated fetal cells (including fetal nucleated red blood cells, white blood cells, and trophoblasts) can be isolated around eight weeks of gestation. Nucleated fetal cells can be isolated using a variety of methods, including fluorescence-assisted flow cytometry (FACS) using scattering and surface labeling, magnetic-mediated cell sorting, microdissection, cell size sorting devices (e.g., microfluidic chips), and cell density sorting methods (e.g., centrifugation).

Nucleic acids, such as DNA, can be obtained from nucleated fetal cells by the following method: Single cells are placed in 30 μl of freshly prepared cell lysis buffer (30 mM Tris-Cl pH 7.8, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100, 12.5 μg/ml QIAGEN Protease). See, for example, Bianchi et al., Isolation of fetal DNA from nucleated erythrocytes in maternal blood, Proc. Natl. Acad. Sci. USA Vol. 87, pp. 3279-3283, May 1990, and Wachtel et al., Clin. Genet. 2001: 59; 74-79 (2001), which are incorporated herein by reference. Other methods, such as alkaline lysis or thaw-freeze lysis, can also be used to extract nucleic acids, or other methods described herein or known in the art.

The extracted nucleic acid can be amplified using the following method. Add 30ul of amplification buffer (20mM Tris-Cl (pH8.8), 10mM (NH4)2SO4, 10mM KCl, 3mM MgSO4, 0.1% Triton X-100, 0.32uM primer GAT3T (GTG AGT GAT GGT TGA GGT AGT GTG GAG NNN NNG GG), 0.25uM primer GAT3G (GTG AGT GAT GGT TGA GGT AGT GTG GAG NNN NNT TT), then heated at 94°C for 3 minutes to melt the double-stranded genomic DNA into single strands. Subsequently, these single-stranded DNAs were quickly placed on ice to ensure their effective binding to the primers. Subsequently, 2.5U of Bst Large Fragment (NEB) or 2U of Bst Large Fragment and 0.8U of Pyrophage 3173 exo- (Lucigen) were added. The following temperature cycle was performed: 10°C-45 seconds, 20°C-45 seconds, 30°C-60 seconds, 40°C-45 seconds, 50°C-45 seconds, 62°C-2 minutes, 95°C-20 seconds, and then the PCR tubes were quickly placed on ice.

After annealing on ice, the same polymerase mixture was added and the following temperature cycle was performed: 10°C for 45 seconds, 20°C for 45 seconds, 30°C for 60 seconds, 40°C for 45 seconds, 50°C for 45 seconds, 62°C for 2 minutes, 95°C for 20 seconds, and 58°C for 20 seconds. The above steps were repeated four times to obtain a mixture with the amplified product.

The amplified product can be further amplified by PCR for next-generation genome sequencing. To the pre-amplified product, 30 μl of freshly prepared amplification mix (20 mM Tris-Cl pH 8.8, 10 mM (NH₄)₂SO₄, 10 mM KCl, 4 mM MgSO₄, 0.1% Triton X-100, 0.66 μM Bio-GAT primer (5'/Biosg/GTGAGTGATGGTTGAGGTAGTGTGGAG-3'), and 2.4 U Deep VentR exo-polymerase (NEB)) was added. The temperature was cycled 18 times: 94°C for 20 seconds, 59°C for 20 seconds, 65°C for 1 minute, and 72°C for 2 minutes. Starting from one cell, a total of 2-3 μg of double-stranded DNA product was obtained for high-throughput sequencing.

The genetic testing performed on the amplicon can be on a whole-genome scale, or on a significant portion of a selected genome, or on a genomic site known to cause disease. Examples of whole-genome analysis include next-generation whole-genome sequencing (Illumina, SoliD, etc.), hybridization-based whole-genome genotyping methods, such as single-base polymorphism arrays (SNP arrays), comparative genomic hybridization chips, and the like. Methods for analyzing significant portions of the genome include targeted sequence resequencing, sequencing of specific genomic portions such as exome sequencing, a certain chromosome, and the like. Examples of analyzing specific genomic sites include hybridization of nucleic acid probes to the amplified whole genome, analysis by imaging or sequencing, and also amplification of specific genomic regions by PCR or multiplex PCR and subsequent sequencing or genotyping.

The range of genetic polymorphisms that are meaningful for prenatal screening and diagnosis is very wide, including but not limited to single base polymorphisms (SNVs), small insertions or deletions (Indels) of 1-100 bp, copy number polymorphisms (CNVs) of 100 bp-100 Mbp, inversions and duplications of sequences of 1 bp-1 Mbp, loss of heterozygosity (LOH) of 10 bp-100 Mbp, and abnormalities at the whole chromosome level, including chromosomal translocations, aneuploidy, and deletion or duplication of part or all of a chromosome.

Examples of "genomically associated disease or known phenotypic outcomes" include consistent diseases associated with genetic mutations, such as beta-thalassemia, which is caused by a 4-bp deletion in the coding regions 41 and 42 of the hemoglobin (HBB) gene, and Down syndrome, which is caused by a duplication of chromosome 21 (trisomy 21). "Consistent phenotypic outcomes" include underlying health conditions or physical conditions that are still considered genetic diseases, such as susceptibility to certain diseases, such as cancer, the sex of the baby, etc. For specific examples, please refer to: Cheung et al., Nature Genetics, Vol. 14, pp. 264-268 (1996) (sickle cell anemia, thalassemia), Belroud, et al., The Lancet, Vol. 361, pp. 1013-1014 (2003) (spinal muscular atrophy)

According to certain other aspects, one or more cells can be cut or separated from the IVF embryo. Nucleic acid, such as DNA, can be extracted from the unicellular or multiple cells in the IVF embryo. These nucleic acid materials are amplified by the amplification method shown here, to carry out analysis of, for example, the entire genome or some part of the genome. Genomic variation or differences in disease-related or known phenotypic outcomes can be detected.

One or more cells from an IVF embryo can be obtained by micromanipulation of the embryo using embryonic puncture. This can involve harvesting somatic cells, trophoblast cells, or blastomere cells, among other options. Biopsies are performed between day 0 and day 6 after fertilization.

Nucleic acids, such as DNA, can be obtained from embryonic cells by the following method: Single cells are placed in 30 μl of freshly prepared cell lysis buffer (30 mM Tris-Cl pH 7.8, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100, 12.5 μg/ml QIAGEN Protease). Other methods, such as alkaline lysis or thaw-freeze lysis, can also be used to extract nucleic acids, or other methods described herein or known in the art.

The extracted nucleic acid can be amplified using the following method. Add 30ul of amplification buffer (20mM Tris-Cl (pH8.8), 10mM (NH4)2SO4, 10mM KCl, 3mM MgSO4, 0.1% Triton X-100, 0.32uM primer GAT3T (GTG AGT GAT GGT TGA GGT AGT GTG GAG NNN NNG GG), 0.25uM primer GAT3G (GTG AGT GAT GGT TGA GGT AGT GTG GAGNNN NNT TT), then heated at 94°C for 3 minutes to melt the double-stranded genomic DNA into single strands. Subsequently, these single-stranded DNAs were quickly placed on ice to ensure their effective binding to the primers. Subsequently, 2.5U of Bst Large Fragment (NEB) or 2U of Bst Large Fragment and 0.8U of Pyrophage 3173 exo- (Lucigen) were added. The following temperature cycle was performed: 10°C-45 seconds, 20°C-45 seconds, 30°C-60 seconds, 40°C-45 seconds, 50°C-45 seconds, 62°C-2 minutes, 95°C-20 seconds, and then the PCR tubes were quickly placed on ice.

After annealing on ice, the same polymerase mixture was added and the following temperature cycle was performed: 10°C for 45 seconds, 20°C for 45 seconds, 30°C for 60 seconds, 40°C for 45 seconds, 50°C for 45 seconds, 62°C for 2 minutes, 95°C for 20 seconds, and 58°C for 20 seconds. The above steps were repeated four times to obtain a mixture with the amplified product.

The amplified product can be further amplified using PCR for next-generation genome sequencing. To the pre-amplified product, 30 μl of freshly prepared amplification mix (20 mM Tris-Cl pH 8.8, 10 mM (NH₄)₂SO₄, 10 mM KCl, 4 mM MgSO₄, 0.1% Triton X-100, 0.66 μM Bio-GAT primer (5'/Biosg/GTGAGTGATGGTTGAGGTAGTGTGGAG-3'), and 2.4 U Deep VentR exo-polymerase (NEB)) was added. The temperature was cycled 18 times: 94°C for 20 seconds, 59°C for 20 seconds, 65°C for 1 minute, and 72°C for 2 minutes. Starting from one cell, a total of 2-3 μg of double-stranded DNA product was obtained for high-throughput sequencing.

The genetic testing performed on the amplicon can be on a whole-genome scale, or on a significant portion of a selected genome, or on a genomic site known to cause disease. Examples of whole-genome analysis include next-generation whole-genome sequencing (Illumina, SoliD, etc.), hybridization-based whole-genome genotyping methods, such as single-base polymorphism arrays (SNP arrays), comparative genomic hybridization chips, and the like. Methods for analyzing significant portions of the genome include targeted sequence resequencing, sequencing of specific genomic portions such as exome sequencing, a certain chromosome, and the like. Examples of analyzing specific genomic sites include hybridization of nucleic acid probes to the amplified whole genome, analysis by imaging or sequencing, and also amplification of specific genomic regions by PCR or multiplex PCR and subsequent sequencing or genotyping.

The range of genetic polymorphisms that are meaningful for prenatal screening and diagnosis is very wide, including but not limited to single base polymorphisms (SNVs), small insertions or deletions (Indels) of 1-100bp, copy number polymorphisms (CNVs) of 100bp-100Mbp, inversions and duplications of sequences of 1bp-1Mbp, loss of heterozygosity (LOH) of 10bp-100Mbp, and abnormalities at the whole chromosome level, including chromosome translocations, aneuploidies, partial or complete deletions or duplications of chromosomes, and other known chromosome or gene variations that may cause disease. It should be noted that the purpose of some of the genetic diseases mentioned here is not to exhaust all diseases, but only to illustrate. The purpose of the method shown here is to analyze the genome of a single cell. Therefore, any or all abnormalities of diseases that can be detected by analyzing the cell genome are considered here as an example of this display method.

Examples of "genomic findings associated with a disease or known phenotype" include consistent diseases associated with genetic variants, such as beta-thalassemia, caused by a 4-bp deletion in the coding regions 41 and 42 of the hemoglobin (HBB) gene, and Down syndrome, caused by a duplication of chromosome 21 (trisomy 21). "Consistent phenotypic findings" also include underlying health conditions or physical conditions that are not yet considered genetic, such as susceptibility to certain diseases, such as cancer, and the sex of the fetus. Other diseases and conditions that can be detected in the fetus or embryo through prenatal testing include cystic fibrosis, sickle cell disease, Tay-Sachs disease, fragile X syndrome, spinal muscular atrophy, hemoglobinopathies, thalassemia, X-linked disorders (diseases caused by genes on the X chromosome), spina schizophrenia, anencephaly, congenital heart disease, obesity, diabetes, cancer, the sex of the fetus, infant RHD, infant HLA typing, paternal mutations, and chromosomal aneuploidy.

Example XVI

Cancer diagnosis

According to certain aspects of the method presented herein, a whole genome analysis of a single, some, or a specific cancer cell is provided. The method presented here is particularly applicable when there are only a small number of cancer cells. For example, when samples are scarce, or when they are scarcely available and can be isolated. A specific example is circulating tumor cells (CTCs) in an individual's blood. During the interaction between tumor cells and blood vessels and during tumor metastasis, cancer cells, such as tumor cells, invade the blood. The current diagnostic method based on circulating tumor cells is based on the counting of enrichable CTC cells. Because CTC cells are very rare in the blood (one CTC in 10^9 blood cells) and CTCs are highly heterogeneous, the efficiency of enrichment varies from case to case. Based on the number of CTCs, it is not very reliable, and this method is now undergoing clinical trials. Currently, CellSearch is the only FDA-approved instrument that can be used for CTC enrichment and counting. Traditional diagnostic methods that use about one million cells to detect genetic diseases may not be applicable to CTCs.

Circulating tumor cells (CTCs) provide a source of cancer cells, either originating in situ or metastatic sites, and can be used to detect and analyze cancer cells for early diagnosis. The method presented here enables DNA detection of CTCs. Because CTCs are so few in number, invasive procedures such as surgery are not necessary for their acquisition, providing a non-invasive method for detection. The method presented here allows for DNA detection of CTCs, potentially enabling detection of cancer cells at their earliest stages, when they are free in the bloodstream. The method described here allows for reliable and uniform amplification of the entire genome of a single cell, as well as of approximately 10 or 100 cells, without introducing excessive amplification bias or allelic dropout. This is because the method can amplify the entire genome or nearly the entire genome of a single cell. This method is particularly useful for the CTCs described here, which are so rare and therefore crucial for early diagnosis.

According to one aspect, the MALBAC method presented herein utilizes multiple annealing and cyclic amplification to amplify the whole genome of a single cancer cell, such as a circulating tumor cell. According to one aspect, circulating tumor cells can be obtained from a patient's blood. Tumor cells can also be obtained from in situ or metastatic cancers through minimally invasive procedures such as microneedle aspiration (FNA) for minimally invasive sampling diagnosis (MSMD). Through these means, obtaining one or more cancer cells can be considered non-invasive. The method presented herein has many applications, particularly in situations where only a few cancer cells can be obtained. The method can also be applied to situations where a larger number of cancer cells can be obtained but single-cell analysis is required.

According to one aspect, a method for analyzing DNA from a cancer cell is provided. "Cancer" refers to various types of malignant proliferation, most of which invade surrounding tissues and may also metastasize to another area (see, for example, the PDRMedical Dictionary, ^1st edition (1995)). The terms "hyperplasia" and "tumor" refer to an abnormal group of cells that continue to proliferate rapidly even after the signaling molecules that stimulate proliferation are removed. These tissues often partially or completely lack the structural and functional components of normal tissues and can be benign (such as benign tumors) or malignant (such as malignant tumors).

Examples of general types of tumors (but not limited to) include: cancer (i.e., malignant tumors, often originating from epidermal tissue, such as common types of breast, prostate, lung, and colon cancer), malignant sarcomas (i.e., malignant tumors originating from connective tissue and mesenchymal tissue; lymphomas, leukemias, and malignant tumors originating from hematopoietic stem cells), germ cell tumors (tumors originating from totipotent cells, generally common in the testicles or ovaries in adults, and common in infants and young children in the midline of the body, especially the tip of the coccyx), blastomas (tumors of immature cells or embryonic tissue), and similar conditions. Those skilled in the art will understand that the examples herein are for illustrative purposes only and are not intended to be exhaustive, and therefore more types of tumors may be detected using the methods presented herein.

Hyperplasia referred to in the method of the present invention includes but is not limited to: acute lymphoblastic leukemia, myeloid leukemia, childhood acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma (cerebellum, cerebrum), atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic hyperplasia, bladder cancer, bone cancer, osteosarcoma, malignant fibrous histiocytoma, brain tumors (such as brainstem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumor, cerebellar astrocytoma, brain astrocytoma/ Malignant gliomas (e.g., craniopharyngioma, ependymoma, medulloblastoma, medulloepithelioma, intermediately differentiated pineal parenchymal tumor, supratentorial primitive neuroectodermal tumor, pinealoblastoma, visual pathway hypothalamic glioma, brain and spinal cord tumors); breast cancer; bronchogenic carcinoma; Burkitt lymphoma; carcinoid tumors (e.g., gastrointestinal), carcinoma of unknown primary; central nervous system (e.g., atypical teratoid/rhabdoid tumors, embryonal tumors (e.g., lymphoma, primary); cerebellar astrocytoma; brain astrocytoma/malignant glioma; cervical cancer; chordoma, chronic lymphocytic leukemia, chronic bone marrow tumors); Myeloproliferative disorders; colon cancer; rectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; embryonal tumors, central nervous system; endometrial cancer; ependymoma; esophageal cancer; Ewing family of tumors; extracranial germ cell tumors; extragonadal germ cell tumors; extrahepatic bile duct cancer; eye cancer (e.g., intraocular melanoma, retinoblastoma); gallbladder cancer; gastric cancer; gastrointestinal tumors (e.g., carcinoid, stromal tumors (gist), interstitial cell tumors); germ cell tumors (e.g., extracranial, extragonadal, ovarian); gestational trophoblastic tumors; gliomas (e.g., brain stem, astrocytomas), hairy cell leukemia, head Neck tumors, liver cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and optic pathway gliomas; intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, renal cancer, large cell tumor, laryngeal cancer (e.g., acute lymphocytic, acute myeloid); leukemias (e.g., acute myeloid, chronic lymphocytic, chronic myeloid, hairy cell); lip and/or oral cancer, liver cancer; lung cancer (e.g., non-small cell, small cell); lymphomas (e.g., AIDS-related, Burkitt's, cutaneous T-cell, Hodgkin lymphoma, non-Hodgkin, primary central nervous system), macroglobulinemia, Waller's disease, bone and/or osteosarcoma malignant fibrous histiocytoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck, oral cancer; multiple endocrine neoplasia syndrome, multiple myeloma/plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic/myeloproliferative disorders, myeloid leukemia (e.g., chronic, acute, multifocal), myeloproliferative disorders, chronic; cancer of the nasal cavity and/or paranasal sinuses; nasopharyngeal carcinoma; neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer; oral cancer; oral cancer, oropharyngeal cancer, osteosarcoma and/or malignant fibrous tissue of the bone ovarian cancer (e.g., epithelial ovarian cancer, ovarian germ cell tumor, ovarian tumor of low malignant potential), pancreatic cancer (e.g., islet cell tumor), papillomatosis, paranasal sinus and/or nasal cavity cancer, thyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pinealoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasmacytoma/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the kidney, renal pelvis and/or ureter, respiratory tract infection Chromosomal gene nut; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcomas (e.g., Ewing family tumors, Kaposi's, soft tissue, uterine); Sézary syndrome; skin cancer (e.g., non-melanoma, melanoma, Merkel cell); small cell lung cancer; small bowel tumors, soft tissue sarcomas, squamous cell carcinoma; squamous cell head and neck cancer with occult primary, metastasis, gastric cancer; supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and/or thymic carcinoma, thyroid cancer, trophoblastic tumor; cancer of unknown primary site, urethral cancer, uterine cancer , endometrial cancer, uterine sarcoma, vaginal cancer, glioma of the visual pathway and/or hypothalamus, transitional cell carcinoma of the kidney, renal pelvis and/or ureter, vulvar cancer; Waldenstrom's macroglobulinemia; Wilms' tumor, etc. For a review, please refer to the National Cancer Institute's global website (cancer.gov/cancertopics/alphalist). One skilled in the art will understand that this list is exemplary only and not exhaustive, as one skilled in the art will readily be able to identify additional cancers and/or tumors based on the disclosure herein.

According to certain aspects, circulating tumor cells are isolated from an individual's blood. One or more circulating tumor cells are then subjected to nucleic acid extraction. The nucleic acids are then amplified using the amplification methods described herein, for example, using multiple annealing cycles with or without subsequent exponential amplification. This provides analysis of the entire genome of the cell or specific genomic loci. The genome is then analyzed for genetic variation and associated genomic diseases and cancers.

Tumor circulating cells can be obtained by the following methods. Blood from the patient is obtained by routine venipuncture. About 10 ml of blood is obtained. Circulating tumor cells can be obtained by a variety of different methods, including: commercial CellSearch system (Clin.Cancer Res.2004,10,6897-6904and Clin.Cancer Res.2010,16,2634-2645), size-based filtration device (J.Pathol.2000,156,57-63and Cancer Res.2010,70,6420-6428), field imaging and fiber optic array scanning technology (Proc.Natl.Acad.Sci.USA2004,101,10501-1050), microfluidic device based on antibody capture (Lab Chip2010,10,837-842,Nature2007,450,1235-1239, Anal.Chem.2011,83,2301-2309, Angew.Chem.2011,123,3140-3144andAngew.Chem.Int.Ed.2011,50,3084-3088)

According to certain other aspects, one or more cells are obtained from a block or tissue mass by fine needle aspiration. Nucleic acids, such as DNA, are extracted from a single cell or multiple cells obtained from fine needle aspiration. Linear pre-amplification is then performed using the methods described herein to obtain the full genome of the embryo or specific genomic loci for analysis. Genetic variations associated with known congenital diseases or with known phenotypes are then determined.

Cells are biopsied and removed by fine needle aspiration as shown below. First, wipe the skin above the area to be examined with disinfectant and sterile surgical towels. After locating the mass, use X-ray or palpation,

A special, very thin (22- or 25-gauge) needle is inserted into the tumor. After the needle is inserted, cells are aspirated with a syringe and transferred to a single tube. The cells are labeled and stored. Individual cancer cells are then isolated using a mouth pipette or laser ablation under a fluorescence microscope.

Nucleic acids, such as DNA, can be extracted from CTCs or fine needle aspirated cells using protease digestion. Single cells are placed in 30 μl of freshly prepared cell lysis buffer (30 mM Tris-Cl pH 7.8, 2 mM EDTA, 20 mM KCl, 0.2% Triton X-100, 12.5 μg/ml QIAGEN Protease). Other methods, such as alkaline lysis or thaw-freeze lysis, can also be used for nucleic acid extraction, or other methods described herein or known in the art.

The extracted nucleic acid can be amplified using the following method. Add 30ul of amplification buffer (20mM Tris-Cl (pH8.8), 10mM (NH4)2SO4, 10mM KCl, 3mM MgSO4, 0.1% Triton X-100, 0.32uM primer GAT3T (GTG AGT GAT GGT TGA GGT AGT GTG GAG NNN NNG GG), 0.25uM primer GAT3G (GTG AGT GAT GGT TGA GGT AGT GTG GAG NNN NNT TT), then heated at 94°C for 3 minutes to melt the double-stranded genomic DNA into single strands. Subsequently, these single-stranded DNAs were quickly placed on ice to ensure their effective binding to the primers. Subsequently, 2.5U of Bst Large Fragment (NEB) or 2U of Bst Large Fragment and 0.8U of Pyrophage 3173 exo- (Lucigen) were added. The following temperature cycle was performed: 10°C-45 seconds, 20°C-45 seconds, 30°C-60 seconds, 40°C-45 seconds, 50°C-45 seconds, 62°C-2 minutes, 95°C-20 seconds, and then the PCR tubes were quickly placed on ice.

After annealing on ice, the same polymerase mixture was added and the following temperature cycle was performed: 10°C for 45 seconds, 20°C for 45 seconds, 30°C for 60 seconds, 40°C for 45 seconds, 50°C for 45 seconds, 62°C for 2 minutes, 95°C for 20 seconds, and 58°C for 20 seconds. The above steps were repeated four times to obtain a mixture with the amplified product.

The amplified product can be further amplified using PCR for next-generation genome sequencing. To the pre-amplified product, 30 μl of freshly prepared amplification mix (20 mM Tris-Cl pH 8.8, 10 mM (NH₄)₂SO₄, 10 mM KCl, 4 mM MgSO₄, 0.1% Triton X-100, 0.66 μM Bio-GAT primer (5'/Biosg/GTGAGTGATGGTTGAGGTAGTGTGGAG-3'), and 2.4 U Deep VentR exo-polymerase (NEB)) was added. The temperature was cycled 18 times: 94°C for 20 seconds, 59°C for 20 seconds, 65°C for 1 minute, and 72°C for 2 minutes. Starting from one cell, a total of 2-3 μg of double-stranded DNA product was obtained for high-throughput sequencing.

The genetic testing performed on the amplicon can be on a whole-genome scale, or on a significant portion of a selected genome, or on a genomic site known to cause disease. Examples of whole-genome analysis include next-generation whole-genome sequencing (Illumina, SoliD, etc.), hybridization-based whole-genome genotyping methods, such as single-base polymorphism arrays (SNP arrays), comparative genomic hybridization chips, and the like. Methods for analyzing significant portions of the genome include targeted sequence resequencing, sequencing of specific genomic portions such as exome sequencing, a certain chromosome, and the like. Examples of analyzing specific genomic sites include hybridization of nucleic acid probes to the amplified whole genome, analysis by imaging or sequencing, and also amplification of specific genomic regions by PCR or multiplex PCR and subsequent sequencing or genotyping.

The range of genetic polymorphisms that are meaningful for cancer diagnosis is very wide, including but not limited to single base polymorphisms (SNVs), small insertions or deletions (Indels) of 1-100bp, copy number polymorphisms (CNVs) of 100bp-100Mbp, inversions and duplications of sequences of 1bp-1Mbp, loss of heterozygosity (LOH) of 10bp-100Mbp, and abnormalities at the whole chromosome level, including chromosome translocations, aneuploidies, partial or complete deletions or duplications of chromosomes, and other known chromosome or gene variations that may cause disease. It should be noted that the purpose of certain genetic diseases mentioned here is not to exhaust all diseases, but only to illustrate. The purpose of the method shown here is to analyze the genome of a single cell. Therefore, any or all abnormalities of diseases that can be detected by analyzing the cell genome are considered here as an example of this display method.

For specific examples of known cancer genomic variants, one can refer to existing public databases such as the Cancer Genome Anatomy Project (CGAP) and the Catalog of Somatic Mutations in Cancer (COSMIC) of the National Institutes of Health.

Claims (28)

1. A method for amplifying the whole genome of a single cell for non-diagnostic purposes, comprising: (a) Providing genomic DNA from a single cell in single-stranded form in a reaction vessel; (b) To produce a reaction mixture, primers having a common sequence of 10-30 nucleotides, a variable sequence of 3-7 nucleotides, and a fixed sequence of 2-4 nucleotides are added to the reaction vessel, wherein the common sequence contains G, T, and A, but not C. (c) The reaction mixture is subjected to a low temperature between 0°C and 10°C, at which the annealing of the primers to the single-stranded genomic DNA occurs. (d) Add at least one DNA polymerase having strand displacement activity or 5'-3' exonuclease activity to the reaction mixture, and subject the reaction mixture to a temperature at which DNA amplification occurs to produce single-stranded or double-stranded DNA. (e) subjecting the reaction mixture to a temperature at which single-stranded amplifiers are produced; (f) subjecting the reaction mixture to an annealing temperature between 55°C and 60°C to the 3' end of the amplifier to maintain a linear structure and prevent the formation of intercalations; and (g) Repeat steps (c) through (f) to produce an amplified version of the genomic DNA. 2. The method according to claim 1, wherein the primers have the following sequences: 5’-GT GAG TGA TGG TTGAGG TAG TGT GGA GNNNNNGGG-3’ and 5’-GT GAG TGA TGG TTG AGG TAG TGT GGAGNNNNNTTT-3’. 3. The method according to claim 1, wherein the DNA polymerase comprises at least one of Φ29 polymerase or Bst polymerase. 4. The method according to claim 1, wherein the DNA polymerase comprises Φ29 polymerase and Bst polymerase. 5. The method according to claim 1, further comprising the step of removing the primer sequences and complementary primer sequences from the 5' and 3' ends of the amplifier. 6. The method according to claim 1, wherein the DNA amplification occurs at a temperature between 30°C and 65°C. 7. The method according to claim 1, wherein the annealing temperature of the free primer is performed before cryogenic quenching. 8. The method of claim 1, wherein step (f) is optional and is performed during the second and subsequent thermal cycles. 9. A method for amplifying a single-cell genome for non-diagnostic purposes, comprising: (a) Providing genomic DNA derived from the single cell in single-stranded form in the reaction vessel; (b) Add primers having a common sequence of 10-30 nucleotides, a variable sequence of 3-7 nucleotides, and a fixed sequence of 2-4 nucleotides to the reaction vessel to generate a reaction mixture, wherein the common sequence contains G, T, and A, but not C; (c) The reaction mixture is subjected to a low temperature between 0°C and 10°C, wherein the primer to the single-stranded genomic DNA annealing occurs at the low temperature; (d) Add at least one DNA polymerase having strand displacement activity or 5’-3’ exonuclease activity to the reaction mixture and subject the reaction mixture to a temperature at which DNA amplification occurs to produce a first-round amplification of single-stranded genomic DNA with primer sequences at the 3’ end; (e) subjecting the reaction mixture to a temperature that decomposes double-stranded nucleic acids into single-stranded nucleic acids; (f) The reaction mixture is subjected to low temperature, at which the annealing of primers to single-stranded genomic DNA and the annealing of primers to the first round of amplifications occur; (g) Add at least one DNA polymerase having strand displacement activity or 5’-3’ exonuclease activity, or a polymerase having 5’ free endonuclease activity, to the reaction mixture, and subject the reaction mixture to a temperature at which DNA amplification occurs to produce a first-round amplified single-stranded genomic DNA with primer sequences at the 3’ end, and a second-round amplified the first-round amplified, the second-round amplified having complementary primer sequences at its 3’ and 5’ ends; (h) subjecting the reaction mixture to a temperature that unwinds double-stranded nucleic acids into single-stranded nucleic acids; (i) The reaction mixture is subjected to a temperature between 55°C and 60°C to anneal the free primers to the 3' end of the second round amplification to maintain a linear structure, or to a temperature between 55°C and 60°C to anneal the 3' end of the second round amplification to its 5' end, thereby forming a ring structure, so that the second round amplification cannot be used for amplification; (j) Repeat (f) to (i) to produce an amplified version of the genomic DNA. 10. The method according to claim 9, wherein the primers have the following sequences: 5’-GT GAG TGA TGGTTG AGG TAG TGT GGA GNNNNNGGG-3’ and 5’-GT GAG TGA TGG TTG AGG TAG TGT GGAGNNNNNTTT-3’. 11. The method according to claim 9, wherein the DNA polymerase comprises at least one of Φ29 polymerase or Bst polymerase. 12. The method according to claim 9, wherein the DNA polymerase comprises Φ29 polymerase and Bst polymerase. 13. The method of claim 9, further comprising the step of removing the primer sequences and complementary sequences at the 5' and 3' ends of the amplifier. 14. The method of claim 9, wherein the DNA amplification occurs at a temperature between 30°C and 65°C. 15. The method according to claim 9, wherein the annealing temperature of the free primer is performed before cryogenic quenching. 16. The method of claim 9, wherein the single cell is a prenatal cell. 17. The method of claim 9, wherein the single cell is a cancer cell. 18. The method of claim 9, wherein the single cell is a circulating tumor cell. 19. A method for amplifying the genome of one or more cells for non-diagnostic purposes, comprising: (a) Providing genomic DNA derived from the one or more cells in single-stranded form in a reaction vessel; (b) Add primers containing a common sequence of 10-30 nucleotides, a variable sequence of 3-7 nucleotides, and a fixed sequence of 2-4 nucleotides to the reaction vessel to generate a reaction mixture, wherein the common sequence contains G, T, and A, but not C; (c) The reaction mixture is subjected to a low temperature between 0°C and 10°C, wherein the primer to the single-stranded genomic DNA is annealed at the low temperature; (d) Add at least one DNA polymerase having strand displacement activity or 5' to 3' exonuclease activity to the reaction mixture, and subject the reaction mixture to a temperature at which DNA amplification occurs to produce a first-round amplification of the single-stranded genomic DNA with primer sequences at the 3' end; (e) subjecting the reaction mixture to a temperature that decomposes double-stranded nucleic acids into single-stranded nucleic acids; (f) The reaction mixture is subjected to low temperature, the annealing of the primers to the single-stranded genomic DNA occurs at the low temperature, and the annealing of the primers to the first round of amplification occurs at the low temperature; (g) Add at least one DNA polymerase having strand displacement activity or 5' to 3' exonuclease activity, or a polymerase having 5' free endonuclease activity, to the reaction mixture, and subject the reaction mixture to a temperature at which DNA amplification occurs to produce a first-round amplification of the single-stranded genomic DNA having primer sequences at the 3' end, and a second-round amplification of the first-round amplification having complementary primer sequences at its 3' and 5' ends; (h) subjecting the reaction mixture to a temperature that unwinds double-stranded nucleic acids into single-stranded nucleic acids; (i) The reaction mixture is subjected to annealing at a temperature between 55°C and 60°C to the 3' end of the second round amplification to maintain a linear structure, or the temperature of annealing the 3' end of the second round amplification to its 5' end is used to form a ring structure, thereby making the second round amplification unusable for amplification; (j) Repeat steps (f) to (i) to produce an amplified version of the genomic DNA. 20. The method of claim 19, wherein the primers have the following sequences: 5’-GT GAG TGA TGGTTG AGG TAG TGT GGA GNNNNNGGG-3’ and 5’-GT GAG TGA TGG TTG AGG TAG TGT GGAGNNNNNTTT-3’. 21. The method of claim 19, wherein the DNA polymerase comprises at least one of Φ29 polymerase or Bst polymerase. 22. The method of claim 19, wherein the DNA polymerase comprises Φ29 polymerase and Bst polymerase. 23. The method of claim 19, further comprising the step of removing the primer sequences and complementary sequences at the 5' and 3' ends of the amplifier. 24. The method of claim 19, wherein the DNA amplification occurs at a temperature between 30°C and 65°C. 25. The method of claim 19, wherein the annealing temperature of the free primer is performed before cryogenic quenching. 26. The method of claim 19, wherein the cell is a prenatal cell. 27. The method of claim 19, wherein the cell is a cancer cell. 28. The method of claim 19, wherein the cell is a circulating tumor cell.