CN1293204C - Allele determination method - Google Patents

Abstract

The present invention provides methods and kits for isolating and identifying alleles, and thus haplotypes, in a genomic DNA sample. The method generally involves hybridizing a primer specific for a polymorphic site in an allele to the DNA sample, extending the primer by one or more nucleic acids, isolating the extended primer, and identifying the allele using the extended primer. The method also allows the ligation of two primers, their isolation and subsequent use in the identification of the target allele. Another primer may also be used in the method as a blocking site for extending the first primer in order to replicate and identify a DNA fragment containing the polymorphic site. The extended or non-extended primers may be labeled so that the primers are easily isolated and/or identified.

Description

Allele determination method

technical field

The present invention relates to methods for isolating and characterizing alleles by identifying one or more heterosequence sites in a gene. More specifically, the invention relates to methods of isolating and identifying alleles using one or more primers.

Background of the invention

The most common form of sequence variation between individuals is single nucleotide polymorphism, commonly known as SNP. As the Human Genome Project was completed, it was estimated that the average number of SNPs present was one in every 1000 nucleotides, but more frequently in certain DNA regions. Efforts are now focused on using SNPs to identify target genes associated with disease or drug response. However, due to the weak correlation, many scientists and researchers doubt the individualized view of drugs and diseases based on individual SNPs, thus the importance of haplotype analysis emerges and becomes a key method for SNP medical applications.

Haplotypes are the organization of individual SNPs along a given stretch of DNA, which is generally known. The classical definition of a haplotype is the combination of alleles at closely linked loci on a single chromosome that tend to be inherited jointly from one generation to the next in a given population. Another aspect of molecular haplotyping is the mapping of linkage disequilibrium, which is now recognized as an important tool in positional cloning of disease genes, and with the genetic dissection of complex phenotypes, many applications become apparent .

Since 1989, scientists have studied various molecular haplotyping methods using single-molecule dilutions (SMDs) of genomic DNA, physically separating alleles, or selecting from heterozygous templates with allele-specific primers Sexually amplified hemizygous DNA fragments for allelic discrimination. However, these methods were only developed for short fragments (approximately 500 bp), but more recently molecular haplotyping has been applied to long-range PCR (another 10-20 fold marker spacing), and the CD4 locus was used as a tool for developing this assay. A prototype system for methods. Other methods of determining DNA sequence haplotypes have also been attempted, but these methods have been largely unsuccessful, unreliable, or costly. Therefore, there remains a need for reliable, high-throughput economical molecular haplotype determination methods.

Summary of the invention

The present invention relates to methods for determining alleles by identifying one or more heterosequence sites in a gene. The method can be used to determine the haplotype of specific genes, and has application value in many fields, including the determination of human leukocyte antigen (HLA) type. The invention also relates to kits for this type of assay.

The present invention includes methods of isolating allele-specific nucleic acid molecules. To a single-stranded nucleic acid molecule comprising one or more heterosequence sites, one or more heterosequence site-specific nucleic acid primers are added and allowed to hybridize. In one embodiment, the 3' end of each primer corresponds to the polymorphic site of the target heterosequence site. In such embodiments, the 3' end can be extended by a single base, ligated to a second primer at the 5' end adjacent to the 3' end of the heterosequence site-specific primer, or can be extended by several base. Extended or linked heterosequence site-specific hybridization primers and nucleic acid molecules are then isolated and, if desired, recovered for further genotyping. In another embodiment, each primer comprises one or more polymorphic bases located within the primer, such that primers that hybridize to less than 100% complementary bases and those that hybridize to 100% complementary bases can be selectively removed. Primers are not affected.

The invention also relates to methods of identifying multiple alleles in a nucleic acid molecule comprising such alleles. A single-stranded nucleic acid molecule containing multiple heterologous sequence sites is selected, and two primers, one heterologous primer and one homologous primer, are added to the nucleic acid molecule. The heterologous primer is capable of hybridizing to a 3' heterosequence site located 3' to the 5' heterosequence site on the same nucleic acid molecule. The base at the 3' end of the heterologous primer corresponds to the polymorphic base at the site of the heterologous sequence, so that extension occurs only when the 3' end of the heterologous primer hybridizes with the single-stranded nucleic acid. The homologous primers are capable of hybridizing to the same nucleic acid molecule at a position 5' to the 5' heterologous sequence site. These primers hybridize to the nucleic acid molecule and extend the heterologous primer so that the 5' heterologous sequence site of the nucleic acid molecule located between the primers is replicated, when the extended heterologous primer reaches the homologous primer, the homologous The primer acts to terminate the extension of the heteroprimer. The nucleic acid molecule and the extended heteroprimer are denatured, and the heteroprimer is isolated and analyzed to determine the 5' heterosequence site. Use this information to determine a new set of nucleic acid primers containing another heterologous primer and another homologous primer, the heterologous primer of the new set of primers can hybridize to the 5' heterologous sequence site (the heterologous primer's The 3' terminal base corresponds to the polymorphic base), the 5' heterologous sequence site is located at the 3' end of another heterologous sequence site on the same nucleic acid molecule, and the homologous primers of this new set of primers can be The 5' end of the other heterosequence site hybridizes to the same nucleic acid molecule. The preceding steps are repeated, using a new set of primers in each subsequent round of hybridization/extension, until sufficient heterosequence sites have been identified on the nucleic acid molecule for allele identification. Haplotypes of nucleic acid molecules can be determined in this manner.

The present invention also relates to a method for identifying multiple alleles in a nucleic acid molecule, comprising adding a nucleic acid sample containing multiple alleles to a set of beads, each bead is attached with two different primers, each bead has at least A primer is a primer for a unique allele such that at least one primer for a unique allele hybridizes to a portion of the nucleic acid sample. The hybridized primer is amplified, and the hybridized primer is extended to produce an extended primer nucleic acid. The hybridized nucleic acid sample and primers are then denatured, and the nucleic acid sample is removed from the beads. The extended primer then hybridizes to a second primer on the bead and amplifies the second primer. The beads containing the dual amplification primers are then analyzed to determine the alleles present in the nucleic acid sample.

Brief description of the drawings

Figure 1 is a schematic diagram illustrating the application of the allele-specific primer extension method of the present invention to identify alleles.

Fig. 2 is a schematic diagram illustrating a single base extension multi-allelic identification method using a primer size marker method.

Figure 2A is a schematic diagram illustrating a single base extension multi-allelic identification method using a primer size marker method.

Figure 3 is a schematic diagram illustrating the identification of alleles using the allele-specific ligation and primer size markers of the present invention.

Figure 4 is a schematic diagram illustrating the use of hybridization and primer size markers of the present invention to identify alleles.

Fig. 5 is a schematic diagram illustrating a multi-allelic identification method using a set of homologous primers and heterologous primers of the present invention.

6A-6F are schematic diagrams illustrating a method for identifying multiple alleles using fluorescent beads comprising multiple primers of the present invention.

Detailed description of the invention

The present invention is based on US Provisional Patent Application No. 60/228,994, which is hereby incorporated by reference in its entirety, and relates to methods of determining allelic markers.

The following terms are used throughout this application and are defined below:

Allele: A variant form of a given gene. Such variants include single nucleotide polymorphisms, insertions, inversions, translocations and deletions.

Avidin: A family of proteins functionally defined by their ability to bind biotin specifically with high affinity. Avidin is a relatively small oligomeric protein composed of four identical subunits, each with a single biotin-binding site. Thus 4 moles of biotin are bound per mole of avidin. Avidin includes (a) a protein produced by amphibians, reptiles, and birds that is present in their eggs called avidin, and (b) the Streptomyces genus Streptomyces avidinus ( Streptomycesavidinii), called streptavidin. "Avidin" as used herein includes all of the above proteins.

Biotin: "Biotin" as used herein includes biotin, commercial biotin products in which biotin is modified by the addition of an alkyl group, and biotin derivatives such as active esters, amines, hydrazides and sulfhydryls, which are found in Additional reactive groups such as amines, acyl and alkyl leaving groups, carbonyl and alkyl halides or Michel-type acceptors are present on the polymer.

Detection Molecule: A molecule covalently attached to a nucleic acid that renders the nucleic acid detectable and/or removable, typically by an external energy source. Such molecules can include dyes, variable weight molecules (including poly-A and poly-T tails), linkers that can be attached to beads (including magnetic beads), biotin, avidin, dihydrogen Octagenin, digoxigenin antibodies, and other similar materials known in the art.

Genotype: The specific alleles carried at a genetic locus.

Haplotype: Represents the collective genotype of several closely linked loci, the complete sequence of alleles on the same chromosome.

Heteroprimer: A primer that hybridizes to a unique allele under stringent conditions.

Heterosequence site: Two alleles that have different sequences at a defined sequence site are said to have a heterosequence site.

Homologous Primer: A primer that hybridizes to alleles from both parents.

Parental Allele: An allele in a diploid mammalian cell that contains one set of chromosomes from the maternal side and one set from the paternal side.

Primer: An oligonucleotide capable of hybridizing to a DNA template.

All patent documents and references cited herein are hereby incorporated by reference.

The method of the present invention has several important advantages. The method of the invention can quickly, low-cost, and accurately determine alleles, including complete genotype and haplotype determination. The length of nucleic acid that can be analyzed by this method is the length of nucleic acid fragments that cannot be fully amplified by standard amplification methods known in the art (eg, polymerase chain reaction).

The present invention relates to methods of isolating and characterizing allele-specific nucleic acid molecules. Any nucleic acid molecule may be used, preferably deoxyribonucleic acid. Allele-specific nucleic acid molecules that can be identified and isolated include alleles, gene segments, and non-expressed segments of polyallelic genes.

The methods and kits of the invention can be used with all diploid genetic material having two or more heterosequence sites and thus multiple allelic types. Examples of genes with multiple alleles to which the present invention can be applied are mammalian MHC genes such as human leukocyte antigen (HLA) class I and class II genes, mammalian T cell receptor genes, TAP, LMP, ras, SARS Type HLA class I genes, genes for human complement factors C4 and C2, Bf in the human HLA complex, and genes located in mitochondrial DNA, bacterial chromosomes, and viral DNA.

In one method of the invention, a nucleic acid sample is obtained that contains multiple alleles, each allele carrying a unique set of heterosequence loci. The nucleic acid sample is amplified by any method known in the art, one embodiment is by polymerase chain reaction (PCR), as described in US Patent No. 4,683,202, published July 28, 1988, to Mullis. The amplified nucleic acid sample is then denatured to single-stranded nucleic acid. This single-stranded nucleic acid can then be analyzed to determine the presence of alleles by determining the presence of heterosequence sites by various methods of the invention.

The methods of the invention use one or more primers. The primers of the present invention comprise a nucleotide sequence that hybridizes to a target sequence. In some cases, it is desirable for primers to hybridize under conditions that enable extension during amplification. In other cases, it is desired that primers that hybridize to 100% complementary match have a higher Tm than primers that hybridize to less than 100% complementary match. In general, primers of the invention may be of any effective length, but generally comprise about 12-25 nucleotides or at least 18 nucleotides, with a preferred length of about 18-22 nucleotides. In the methods of the invention, it is necessary to identify one or more primer sequences unique to the target DNA in the sample in order to identify polymorphic sites of the sequence of interest. Such polymorphism identification of many multi-allelic genes is well known in the art. For example, the HLA-A, HLA-B, and HLA-C genes have approximately 222 known alleles, the sequences of which are well known in the art. See Arnett and Parham, Tissue Antigens 45:217-257, 1995, and US Patent No. 5,702,885, published December 30, 1997, to Baxter-Lowe et al.

As used herein, the phrase "hybridizes under highly stringent conditions" to describe hybridization of nucleic acid molecules refers to hybridization under conditions of low ionic strength and high wash temperatures. The phrase "hybridizes under low stringency conditions" refers to hybridization under conditions of high ionic strength and low temperature.

Variables that affect stringency include, for example, temperature, salt concentration, probe/sample homology, and wash conditions. Stringency increases as the hybridization temperature increases, other things being equal. Increased stringency results in less non-specific hybridization, ie less background interference. "Highly stringent conditions" and "moderately stringent conditions" for nucleic acid hybridization are explained in Current Protocols in Molecular Biology, Ausubel et al., 1998, Green Publishing Associates and Wiley Interscience, NY, the teachings of which are incorporated by reference. Of course, the skilled artisan understands that the stringency of hybridization conditions can be varied as necessary to include or exclude different degrees of complementarity between probe and analyte, and to achieve the desired detection range.

A wide variety of detection molecules can be used in the present invention. These molecules can be coupled to one or more primers, or can be coupled directly to ddNTPs that are inserted into the nucleic acid during the extension step. These molecules may contain means to detect said molecules, such as dyes, radiolabels, etc.; or they may contain means to isolate said molecules, such as biotin/avidin, magnetic and/or fluorescent beads, etc.; or both . For example, when using biotin/avidin, one or more primers can be labeled with biotin so that when the primers hybridize to a single-stranded nucleic acid, one strand of the resulting double-stranded DNA is biotin-labeled. The double-stranded DNA can then be bound to a solid support coated with avidin.

The solid phase carrier used in the present invention can be any such carrier known in the art, such as beads, affinity chromatography column. A preferred carrier is in the form of magnetic beads. When the support is in the form of beads, the two strands of the amplified nucleic acid are separated by attracting the beads with a magnet and washing the beads under conditions that dissociate the double-stranded nucleic acid into single-stranded nucleic acid. Usually under alkaline conditions, typically 0.1M or 0.15M NaOH, the dissociation is performed by incubating the beads several times for about 5-10 minutes at room temperature. Individual strands can then be recovered and further analyzed.

Alleles are determined using a variety of analytical techniques to identify isolated heterosequence sites. These techniques are well known in the art and include, but are not limited to, electrophoresis such as polyacrylamide gel electrophoresis, flow cytometry, high pressure liquid chromatography, laser scanning, and mass spectrometry. These techniques can be performed manually or by automated systems. Such automated systems are well known in the art and include automated sequencers or capillary electrophoresis machines capable of scanning fluorescence of multiple colors.

The first method of the present invention, illustrated schematically in Figures 1 and 2, relies on the extension of hybridized heterosequence site-specific primers. The method is particularly suitable for determining allele- or haplotype-specific genotype information in highly polymorphic chromosomal regions. As shown in Figure 1, after amplification and denaturation of a DNA sample to generate single-stranded DNA fragments, one or more heterologous sequence site-specific primers labeled at the 5' end with a detection molecule are added. A heterosequence site-specific primer is added to a single-stranded nucleic acid molecule and allowed to hybridize. In a preferred embodiment, the 3' end of each primer is complementary to the polymorphic base at the heterologous sequence site. Thus, if the primer hybridizes to a heterologous sequence site where the 3' base is not complementary, the primer will not extend when subjected to extension conditions. Preferably, enzymes that can discriminate between single nucleotide differences are used. As shown in Figure 1, the hybridized primers are then extended, and only primers that hybridize to a complementary 3' base match are extended. The primer is then removed by the detection molecule, illustrated in Figure 1 using biotin as an example. Primers were removed by biotin on the primers using avidin-coated magnetic beads. The hybridized primers/DNA fragments are then washed under conditions such that DNA fragments bound to those unextended primers are removed. The extended double-stranded nucleic acid is then denatured. Strands that are no longer bound to the beads are analyzed to identify heterosequence sites.

Alternatively, the primers used in the present invention may not be coupled to the detection molecule at the 5' end, but instead the primers are hybridized as previously described, using ddNTPs coupled to the detection molecule to single base those primers that hybridize to the complementary 3' end. Base extension, as shown in Figure 2. The primers are isolated using detection molecules on the extension primers, which are then denatured and analyzed to determine the presence of heterosequence sites.

The present invention is also applicable to high-throughput single nucleotide polymorphism measurement using an automatic sequencer capable of scanning fluorescence in four colors or a capillary electrophoresis apparatus using the following method. The same method can also be modified to measure other types of genetic variation besides single nucleotide polymorphisms, including polybasic polymorphisms, insertions, inversions, translocations, and deletions.

Another method of the invention relies on allele-specific ligation. Figure 3 illustrates this approach. As shown in Figure 3, heterosequence site-specific primers are added to single-stranded DNA fragments containing one or more heterosequence sites. The 3' end of each primer of the heterologous sequence-specific primers is complementary to a polymorphic base at a heterologous sequence site, and hybridizes to the DNA fragment. Ligation primers are then added and allowed to hybridize to the DNA fragments. Each ligation primer has a sequence complementary to a portion of one of the DNA fragments such that the 5' end of the ligation primer is directly adjacent to the 3' end of the heterosequence site-specific primer. If the heterosequence site-specific primer does not hybridize to the DNA fragment, then the ligation primer cannot ligate to the heterosequence site-specific primer when subjected to ligation conditions. Primers are ligated, if possible, and then subjected to temperature conditions sufficient to denature unligated primers but insufficient to denature ligated primers that hybridize to the DNA fragment. Typically, such a temperature is about 60°C when a 20 nucleotide primer is used. The hybridized ligation primers can then be removed by any method known in the art. As shown in Figure 3, a set of primers can be attached with a detection molecule, such as biotin. The detection molecule can be attached to a heterologous sequence-specific primer or attached to an adapter primer. Furthermore, the different methods described can be combined, as shown in Figure 3, with one method detecting polymorphisms at one heterosequence site and other methods described herein for detecting polymorphisms at other sites. As also shown in Figure 3, one or more primers can have molecules of variable weight coupled to the 5' end of each primer such that no two primers have the same molecular weight. Such variable weight molecules can be any suitable material that is non-reactive during the hybridization/amplification step, including polyhomotype nucleic acid tails, such as polyadenylic acid tails (poly A). The difference in length of such poly A tails is 2-4 bases, but may be of any different length sufficient to separate poly A tailed primers on standard separation instruments such as gel electrophoresis.

Figure 4 illustrates another method of the present invention. According to such methods, a set of heterosequence-specific primers is added to a DNA fragment containing multiple heterosequence sites. Each primer has at least one polymorphic base, which is located within each primer, such that, after the primers hybridize to the DNA fragment, the Tm of those primers with mismatched bases in the hybridization is higher than that of those primers without mismatched bases. Tm is low. This difference in Tm is then used to remove those primers that hybridize to less than 100% complementarity. Such base mismatches generally occur near the center of the primer sequence. After removal of primer/DNA fragment conjugates that hybridize less than 100% complementary, the remaining conjugates are analyzed to determine specific heterosequence sites and determine specific alleles. This can be done in a variety of ways. According to Figure 4, all primers can be attached to molecules of variable weight. All primers for each specific heterosequence site can be linked to specific variable weight molecules. Each primer for each polymorphism at one or more specific heterosequence sites is linked to a different detection molecule. The hybridized primers are sorted into groups according to the detection molecule, and the allele-specific identity is determined by further determining which variable weight molecule is present in each group of primers.

Another method of the invention enables the determination of multiple heterosequence sites on long fragments of nucleic acid that may be too long to be fully amplified by conventional methods such as PCR. As shown in Figure 5, single-stranded nucleic acid molecules containing multiple heterosequence sites are selected. Two primers, a heterologous primer and a homologous primer, are added to the nucleic acid molecule. A heteroprimer is capable of hybridizing to a 3' heterosequence site located 3' to a 5' heterosequence site on the same nucleic acid molecule. The base at the 3' end of the heterologous primer corresponds to the polymorphic base at the site of the heterologous sequence, so that extension occurs only when the 3' end of the heterologous primer hybridizes with the single-stranded nucleic acid. The homologous primer is capable of hybridizing to the same nucleic acid molecule at a position 5' to the 5' heterologous sequence site. The primers hybridize to the nucleic acid molecule, and the heteroprimers are extended to replicate the 5' heterosequence site of the nucleic acid molecule located between the primers. The nucleic acid molecule and the extended heteroprimer are denatured, and the heteroprimer is isolated and analyzed to determine the 5' heterosequence site. This information is used to identify a new set of nucleic acid primers containing a heterologous primer and a homologous primer, the heterologous primer of the new set of primers hybridizes to the 5' heterologous sequence site (the 3' end of the heterologous primer The base corresponds to the polymorphic base), the 5' heterologous sequence site is located at the 3' end of another heterologous sequence site on the same nucleic acid molecule, and the homologous primer of this new set of primers can be combined with the position The same nucleic acid molecule hybridizes at a position 5' to another heterosequence site. The preceding steps are repeated, using a new set of primers in each subsequent round of hybridization/extension, until a sufficient number of heterosequence sites on the nucleic acid molecule for allele identification are identified. Haplotypes of nucleic acid molecules can be determined in this manner.

As shown in Figures 6A-6F, the present invention also relates to methods of identifying multiple alleles in a nucleic acid molecule. As shown in Figure 6A, the method includes adding a nucleic acid sample containing multiple alleles to a set of beads, each of which is linked with two different primers, at least one primer on each bead is for one Primers for unique alleles. The nucleic acid is then reacted such that at least one primer for a unique allele hybridizes to a portion of the nucleic acid sample, as shown in FIG. 6B. The hybridized primer is amplified to extend the hybridized primer to produce an extended primer nucleic acid, as shown in Figure 6C. Referring to Figure 6D, the hybridized nucleic acid sample and primers are then denatured and the nucleic acid sample is removed from the beads. The extended primer then hybridizes to a second primer on the bead (6E) and amplifies the second primer (6F). The beads containing the dual amplification primers are then analyzed to determine the alleles present in the nucleic acid sample. The primers may have cleavage sites for easy removal from the beads.

The invention also relates to kits for carrying out the methods of the invention. In most basic embodiments, the kits of the invention comprise instructions for carrying out the methods described above. In addition, the kit can contain at least one or several necessary reagents used in the method of the present invention, such as one or several sets of site-specific amplification primers, polymerase chain reaction buffer, dideoxynucleotide (wherein One or more are labeled as required), nucleic acid amplification reagents, reagents for generating single-stranded nucleic acid fragments, one or more heterologous sequence site-specific primers (coupled with at least one detection molecule as required ), one or more ligation primers, reagents for ligation of proximity hybridization primers, beads comprising one or more detection molecules, and one or more sterile microtubes.

The invention will be better understood from the following examples. However, those skilled in the art will easily understand that the specific methods and results discussed are just to illustrate the present invention rather than limit the present invention.

Example

This example involves the application of three strategies to validate the capture of distinct alleles associated with specific polymorphisms in HLA genes: i) hybridization; ii) single base extension; and iii) ligation .

Each of these conditions was used as an assay to establish a test method useful for identifying the appropriate allele and thus the particular polymorphism associated with that allele. The latter two methods are enzyme-based tests that require the use of Taq ligase and Thermus sequencing enzymes and exploit the ability of these enzymes to discriminate single nucleotide differences at specific sites on single-stranded DNA. These methods have been observed to be sufficiently sensitive to resolve single nucleotide polymorphisms or mutations in the particular alleles under study.

1. A. Hybridization

One detection method is hybridization of specific captured targets to oligonucleotide-conjugated microspheres and analysis of the complex. The reaction was carried out as described below. Two rounds of hybridization were performed for the alleles to be captured. The first round of hybridization uses different homozygous and hybrid DNA coupled to beads with specific oligonucleotides that recognize specific sequences. The second round of hybridization, using another set of beads that recognize specific sequences in the target, confirms the presence of the captured allele. However, a single round of hybridization was performed first as a control experiment to determine the specificity of the oligonucleotide-conjugated beads for different alleles in the target.

Using various genomic DNA samples obtained from the UCLA Registry (UCLA 210, UCLA 230 and UCLA 243), 158 bp DNA of the HLA-A locus was amplified using the sense primer 5′A200A and the antisense primer 3′A322-1 fragment. For this example, the 158 bp fragment was prepared using standard amplification methods. The primers used to amplify homozygous DNA and heterozygous DNA in this example are: 5'A200A 5'-ACA GCG ACG CCG CGA GCC A-3', position 182-200, sense primer 3'A322-1 5'-CCTCGCTCTGGTTGTAGTA-3', position 322-340, antisense primer

Single-stranded DNA (ss) for ligation, single-base extension, or hybridization is prepared by asymmetric PCR. The conditions for asymmetric PCR were as above except that the sense primer was added at a concentration 50-fold lower than the antisense primer. The antisense primer is biotinylated, resulting in a 5' biotin-labeled single-stranded PCR fragment.

Alternatively, prepare ssDNA using 5'-3' exonuclease, T7 gene 6 exonuclease. In this case, the strand of interest was protected during oligonucleotide synthesis by introducing a 4-phosphorothioate bond at the 5' end of the PCR primer. T7 exonuclease degrades the 5′ end of the primer that does not contain phosphorothioate bases

1.B. Single base extension reaction (SBER)

The single base extension reaction (SBER) in this example uses an extension primer designed so that its 3' end anneals adjacent to the polymorphic base. The extension method of this example uses Thermo Sequenase or Klenow large fragment polymerase to introduce polymorphic bases in cyclic or acyclic reactions, respectively.

A single base extension reaction was used in an attempt to capture specific alleles; allele-specific PCR (ASPCR) was performed using primer mix (PM) H001 and H002. Use these two primer mixes to introduce specific bases at polymorphic sites. Both PMs used a common 5' primer (agcgacgccgcgagcca), but an allele-specific 3' primer. When using hybrid DNA, PM H001 specifically introduces "C" (ccaagagcgcaggtcctcg) base at the corresponding polymorphic site, while PM H002 specifically introduces "A" (ccaagagcgcaggtcctct).

The extension reaction was performed as described above. Products from extension reactions were purified and bound to streptavidin magnetic beads. The high binding affinity of streptavidin for biotin allows rapid and efficient isolation of biotin-labeled target molecules. The complexes were washed several times to reduce the possibility of any unbound label that could affect the next step in the experiment.

Multiple different samples were tested to verify captured alleles by ASPCR. Five ASPCRs were performed using PMH001 and H002. The experimental conditions and negative controls of a typical extension reaction method are as follows: the experimental sample used in the extension reaction is biotinylated A or C. Assuming that the sequencer will correctly introduce the specific bases, the correct signal from the captured specific allele will be detected based on the primer mix used. The two negative controls and the experimental samples in SBER have the same composition except that ddNTPs A or C were removed from the reaction. Another negative control uses only unlabeled ddNTPs A, C, G and T. The supernatants of the following sets of reactions were verified by ASPCR using primer mixes H001 and H002. The tested supernatants were divided into 5 groups as follows: after extension, after binding of extension products to magnetic beads, after several washes, and after elution of products from magnetic beads at high temperature.

cyclic reaction

Use 100 ng of single-stranded (ss) DNA of the HLA A locus per 20 μl reaction, obtained after PCR amplification of genomic DNA as described above; add 2 μM extension primer, 125 nM each unlabeled dideoxy terminator to the reaction mixture (ddG, T, A or C), and 500nM biotin-labeled ddNTP (A or C, depending on the specific base to be introduced at the polymorphic site), 10X enzyme reaction buffer (diluted to 1X final concentration) and 5 units of Sequenase. The reaction was performed at 94°C for 1 minute, followed by 40 cycles of 94°C for 10 seconds and 60°C for 30 seconds. A final extension cycle at 72°C for 10 minutes and a hold at 4°C was the extension reaction in this example.

acyclic reaction

When extension is performed using the Klenow large fragment polymerase reaction, the first step requires the extension primer to hybridize to single-stranded DNA. 100 ng of ssDNA annealed to 20 μM of the extension primer. The primers and DNA were mixed at 90°C for 5 minutes and then slowly cooled to room temperature, thus generating a hybrid. This process lasts about 1 hour. The next step consists of adding specific unlabeled and labeled biotin ddNTPs (1.5 μM) and 5 U Klenow large fragment and incubating at 37°C for 30 minutes. After extension, 1.5 μl of 0.5M EDTA was added to the reaction mixture.

Extension products (circulating or non-circulating) were purified using QIAQUICK(R) columns (Qiagen) to remove unincorporated biotin. 10 μl of streptavidin-coated magnetic beads (in 2X binding buffer 10 mM Tris pH 7.5, 1 mM EDTA, 2.0 mM NaCl) were mixed with 20 μl of purified extension products for 20 min at room temperature. Apply a magnetic field to the beads to discard unbound extension products. The beads were washed at least twice with 1 ml of the same binding buffer, and the target strand was eluted from the beads by heating at 95°C for 2 minutes.

The eluted strand is then subjected to allele-specific PCR (ASPCR) using specific primers to confirm the polymorphism of the specific allele. Appropriate control experiments were also performed to verify the results.

1.C. Connection method

This example involves the use of a ligation between two primers prior to annealing to a single-stranded DNA template. The implementation of this example is based on the understanding that ligation of two primers that perfectly match ssDNA will generate a strong duplex and thus tolerate higher temperature washes (above the Tm of each primer). Mismatched templates, on the other hand, will hardly tolerate washing at temperatures above the Tm of the primers, and thus themselves will dissociate from the duplex and eventually be washed away.

Two primers are placed adjacent to each other, one of which is an allele-specific or heterosequence primer with a polymorphic site at the 3' end and a biotin tag at the 5' end. The second primer is the ligation primer, which has a phosphate group at the 5' end to mediate ligation. It is assumed that the two primers are ligated together prior to hybridization to the ssDNA template, but the method of the invention does not rely on this assumption. A 20 μl reaction mixture contained 10 μl (100 ng) of specific ssDNA, 1 μl of each primer (1 μM), 2 μl of 10X ligation buffer, and 10 U of Taq ligase.

The mixture was heated in a thermal cycler at 90°C for 2 minutes, followed by incubation at 37°C for 30 minutes, at which time the reaction was terminated by the addition of EDTA. The mixture was purified using a QIAQUICK ^(R) column to remove all unbound primers and biotin, which are non-specific factors in the allele-specific PCR reaction.

Purified complexes were bound to streptavidin-coated magnetic beads as described above. The complexes are washed under high stringency wash conditions. The stringency of the wash is controlled by increasing the temperature of the wash buffer (55-95° C.) in order to achieve the threshold temperature for the isolation of allele-specific DNA fragments. The eluted template was further validated by allele-specific PCR using primers that recognize the polymorphic site of the captured allele.

2. Hybridization method for haplotype determination

Different oligonucleotides for HLA A locus-specific polymorphisms were coupled to different sets of beads (Luminex) used in the hybridization method. Templates for hybridization to oligonucleotide-conjugated beads are chosen to provide complete sequence homology. Coupling of beads to specific oligonucleotides was performed according to the manufacturer's instructions (Luminex Corp.). Luminex bead-probe conjugates were hybridized to the PCR fragment prepared above. The probe sequences used to isolate allele-specific PCR fragments are:

L5′A107A 1AGGTATTTCT A CACCTCCGTG

L5′A107C 1AGGTATTTCT C CACATCCGTG

Non-hybridized PCR templates were washed away, and PCR fragments that specifically hybridized to 5'A107A or 5'A107C were eluted from the Luminex beads. Oligonucleotides of different sizes with and without a spacer (i.e., containing an additional 20 random bases in the middle of the oligonucleotide sequence), coupled to different sets of beads, and hybridized to different templates, determine different alleles gene specificity. The numbers in the primer designations correlate to the different oligonucleotides coupled to the beads and indicate the polymorphic site for the specific allele. For example, 107A or C indicates that the polymorphic site is at base 107, where each allele has an A or C at position 107.

The hybridization reaction procedure was as follows: 17 μl of ssDNA was denatured at 95°C for 5 minutes, followed by the addition of 33 μl of specific oligonucleotide-coupled beads (5000 beads/each oligonucleotide) (complementary to the template), and Incubate at 55°C for 30 minutes. When using oligonucleotides with spacers, the hybridization temperature was increased to 65°C to ensure specificity. The bead mixture was vortexed well and sonicated to bring to the required hybridization temperature before adding ssDNA. After hybridization, the mixture was centrifuged at 2000xg; washed twice with 1.5X TMAC (3M TMAC, 0.1% SDS, 50mM Tris-Cl, pH 8.0, 4mM EDTA pH 8.0), 1ml each time, and the supernatant was discarded .

20 μl of H ₂ O was added to the complex, and the capture template bound to the oligonucleotide-conjugated magnetic beads was eluted at 95° C. for 5 minutes. Asymmetric PCR was performed on 1 μl of the eluted template to obtain a more abundant eluted template for the second round of hybridization.

A second round of hybridization was performed with a second set of beads complementary to the capture template as a test to verify the accuracy of the template. Samples were assayed on a Luminex 100 flow cytometer after addition of 120 ng streptavidin-phycoerythrin (SA-PE) to each tube and incubation at hybridization temperature for an additional 5 minutes. The amount of fluorescent signal obtained is a true representation of the interaction between biotin and SA-PE. The method is a quantitative assay, with the amount of positive signal expressed as the maximum number obtained for a given reaction.

Additional allele-specific Luminex bead-probes used in the second round of hybridization were as follows:

Luminex Bead-Probes for Confirmation of Allele-Specific Segregation:

L5′A107A 1AGGTATTTCTACACCTCCGTG

L5′A107C 1AGGTATTTCTCCACATCCGTG

L5′A153A 1CTTCATCGCAGTGGGCTAC

L5′A153C 1CTTCATCGCCGTGGGCTAC

L5′A249T 1GCAGGAGGGTCCGGAGTAT

L5′A249G 1GCAGGAGGGGCCGGAGTAT

L5′A291C 1GAAGGCCCACTCACAGACT

L5′A291G 1GAAGGCCCA G TCACAGACT

Table 1. Expected Allele-Specific Response Patterns Following Hybridization Template DNA name Luminex Bead-Probe Reaction Mode HLA-A allele L5'A107A L5'A107C L5'A249G L5'A249T L5'A291C L5'A291G UCLA 210 (homozygous) A*0206,- + - - + + - UCLA 230 (heterozygote) A*2402101 + + + - + - A*3401 + - + - - + UCLA 243 (homozygous) A*2402101, - - + + - + -

Table 2. Observed Allele-Specific Response Patterns Following Hybridization Template DNA name probe L5'A107A L5'A107C L5'A249G L5'A249T L5'A291C L5'A291G UCLA 210 (homozygous) L5'A107A (+)166 (-)50 (-)124 (+)279 (+)234 (-)twenty one L5'A107C (-)152 (-)60 (-)137 (-)330 (-)223 (-)29 UCLA 230 (heterozygote) L5'A107A (+)63 (+)111 (+)90 (-)56 (+)94 (-)27 L5'A107C (-)52 (+)87 (+)70 (-)55 (+)57 (-)13 UCLA 243 (homozygous) L5'A107A (-)13 (-)57 (-)37 (-)twenty three (+)96 (-)14 L5'A107C (-)15 (+)83 (+)60 (-)36 (+)124 (-)13 negative control 7 9 14 19 6 9

Table 3. Allele Specific Response Patterns After Hybridization Observed Using Negative Controls Template DNA name no probe (control) L5'A107A L5'A107C L5'A249G L5'A249T L5'A291C L5'A291G UCLA 210 (homozygous) (+)65 (-)29 (-)65 (+)124 (+)97 (-)19 UCLA 230 (heterozygote) (+)63 (+)111 (+)90 (-)56 (+)30 (-)68 UCLA 243 (homozygous) (-)12 (-)216 (-)100 (-)twenty three (+)213 (-)10 negative control 7 9 14 19 6 9

The results in the table above demonstrate successful allele-specific crosses, as the allele-specific numbers are larger than the non-allele-specific responses.

As will be apparent to those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein include any and all possible subranges and combinations thereof. It is readily understood that all listed ranges are a full disclosure and description of the same range divided into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, the ranges discussed here are readily divided into lower thirds, middle thirds, upper thirds, and the like. It will also be understood by those skilled in the art that all descriptions such as "at most," "at least," "greater than," "less than," etc. refer to ranges that may then be divided into sub-ranges as described above.

Although only a few preferred embodiments have been described, it will be apparent to those skilled in the art that modifications and changes may be made in the embodiments without departing from the true spirit and scope of the invention. Accordingly, the preferred embodiments described above are in all respects illustrative and not restrictive of the scope of the invention which is indicated by the appended claims rather than by the foregoing description and which falls within the meaning of the claims. All changes within and within the equivalent range are intended to be embraced within the scope of the invention.

The following references are incorporated by reference in their entirety in this patent application:

Jorde, L.B.: Am.J.Hum.Genet. 56, pp.11-14, 1995;

Thomson, G.: American Journal of Human Genetics (Am.J.Hum.Genet.) 57, pp.474-486, 1995;

Ruano, G., Kidd, K.K. and Stephens, J.C.: Proc. Natl. Acad. Sci. USA 87, pp. 6296-6300, 1990;

Ruano, G. and Kidd, K.K.: Nucleic Acids Res. 19, pp.6877-6882, 1991;

Beloin, S.M., Tishkoff, S.A., Bentley, K.L., Kidd, K.K. and Ruano, G.: Nucleic Acids Res. 24, pp.4841-4843, 1996;

Gilles, P.N., Wu, D.J., Foster, C.B., Dillon, P.J., and Chanock, S.J.: Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor chips microchips), Nature Biotechnology (NatureBiotechnol.) 17, pp.365-370, 1999;

Little, D.P., Braun, A., O'Donnell, M.J., and Koster, H.: Mass spectrometry from miniaturized arrays for full comparative DNA analysis, Nature Med. 3, pp.357-362, 1997;

Marshal, R.D., Koonts, J., and Sklar, J.: Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases by bacteriophage resolvases, Nature Genet. 9, pp.177-183, 1995;

Nauck, M.S., Gierens, H., Nauck, M.A., Marz, W., and Wieland, H.: Rapid genotyping of human platelet antigen 1 (HPA-1) by hybridization probes labeled with fluorophores on a photocycler Determination (Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the Lightcycler), Brit.J.Haematol.105, pp.803-810, 1999;

Pease, A.C., Solas, D., Sullivan, E.J., Cronin, M.T., Holmes, C.P., and Fodor, S.P.A.: Light-generated oligonucleotide arrays for rapid DNAsequence analysis ), Proc.Natl.Acad.Sci.USA, 1994;

Southern, E.M.: DNA chips: large-scale hybridization with oligonucleotides for sequence analysis (DNA chips: Analysis sequence by hybridization tooligonucleotides on a large scale), Genetics Dynamics (Trends Genet.) 12, pp.110-115, 1996 ;

Syvanen, A.C., Aalto-Setala, K., Harju, L., Kontula, K., and Soderlund, H.: A primer-guided nucleotide incorporation assay in apolipoprotein E genotyping assay in the genotyping of apolipoprotein E), Genome (Genomics) 8, pp.684-692, 1990;

Tyagi, S. and Kramer, F.R.: Molecular beacons: Probes that fluoresce upon hybridization, Nature Biotechnol, 14, pp.303-308, 1996.

Sequence Listing

<110> Liu Xiangjun

<120> Allele Determination Method

<130>034928/0112

<140> US 09/943,416

<141>2001-08-30

<150>US 60/228,994

<151>2000-08-30

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Claims (12)

1. A method for detecting alleles by hybridization, comprising: Complexes are formed by hybridizing the target oligonucleotide to oligonucleotides coupled to different sets of beads, wherein the oligonucleotides coupled to different sets of beads are oligonucleotides with and without spacers glycosides; and The complex is analyzed to determine the specificity of the different alleles. 2. The method of claim 1, further comprising isolating allele-specific nucleic acid fragments. 3. The method of claim 2, wherein said isolating allele-specific nucleic acid fragments comprises using polymorphic-specific oligonucleotides coupled to different sets of beads. 4. The method of any one of claims 1-3, further comprising coupling polymorphism-specific oligonucleotides to different sets of beads. 5. The method of any one of claims 1-4, further comprising coupling the oligonucleotides with and without spacers to different sets of beads. 6. The method of any one of claims 1-5, further comprising obtaining a target nucleic acid sample comprising a plurality of alleles, each allele having a unique set of heterosequence sites. 7. The method of any one of claims 1-6, further comprising amplifying the target nucleic acid sample. 8. The method of any one of claims 1-7, further comprising denaturing the target nucleic acid to a single-stranded nucleic acid. 9. The method of any one of claims 1-8, further comprising verifying the target oligonucleotide by hybridizing the target oligonucleotide to a second set of beads and measuring the hybridization of the target oligonucleotide by flow cytometry. The sequence of the target oligonucleotide, wherein the second set of beads is complementary to the target oligonucleotide. 10. The method of any one of claims 1-9, wherein the target oligonucleotide is an HLA allele. 11. The method of any one of claims 1-10, wherein said different sets of beads coupled to oligonucleotides with and without a spacer are conjugated to different oligonucleotides and can be incorporated into a Fluorescent colorimetric identification in one or more beads of the set of beads. 12. A composition comprising oligonucleotides coupled to different sets of beads, wherein the oligonucleotides coupled to different sets of beads are oligonucleotides with and without spacers.

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