HK1230651B - Probe set for analyzing a dna sample and method for using the same - Google Patents

Description

Probe sets for analyzing DNA samples and methods of using the same

Cross-references

This application claims the benefit of provisional application serial number 62/220,746, filed September 18, 2015, which is incorporated herein by reference in its entirety.

background

Cell-free DNA ("cfDNA") can be analyzed to provide prognosis, diagnosis, or predict response to treatment for a variety of diseases and conditions, including various cancers, transplant failure or success, inflammatory diseases, infectious diseases, and fetal aneuploidy.

Cell-free fetal DNA (cffDNA) is present in the blood of pregnant women. This discovery results in the possibility of using a blood sample from a pregnant woman to carry out non-invasive prenatal testing (NIPT) of the fetus. Non-invasive prenatal testing (e.g., amniocentesis or chorionic villus sampling (CVS)) may cause pressure on the mother and some people think that this type of surgery may increase the risk of miscarriage. NIPT can provide information related to multiple genetic defects (including Down syndrome (trisomy 21), Patau syndrome (trisomy 13) and Edward syndrome (trisomy 18)). This type of method should be highly robust because false positives can lead to unnecessary medical procedures, and false negatives may deprive expectant mothers of their understanding of available medical options.

There are many technical obstacles associated with performing non-invasive prenatal testing on a clinical scale. For example, many NIPT efforts have been devoted to analyzing cffDNA to identify copy number changes of specific sequences (e.g., sequences from chromosome 21). However, this type of method is difficult to perform in a robust manner, in part because the vast majority of cfDNA in a blood sample is maternally derived and in many cases only a very small amount (e.g., an average of about 10% and as low as about 3%) is from the fetus. For example, the presence or absence of an extra copy of a chromosome (such as chromosome 21) in the fetus can be determined by comparing the copy number of the sequence corresponding to chromosome 21 with the copy number of the sequence corresponding to an autosome. Although this type of method sounds attractive, it is actually challenging because the fractional concentration of fetal DNA relative to maternal DNA in maternal blood may be as low as 3%. Thus, for every 1000 sequences corresponding to chromosome 21 present in the maternal bloodstream, only a small percentage of these sequences (e.g., if the fetal fraction is 3%, then 30 sequences) are from the fetus. Therefore, an extra copy of a chromosome in the fetus will only result in a relatively small increase in the number of sequences corresponding to that chromosome in the maternal bloodstream. For example, if the fetal fraction is 4, fetal trisomy 21 will only result in a 1.5% increase in the number of fragments corresponding to chromosome 21 in the maternal bloodstream. Due to this problem, statistical rigor can only be achieved by counting a large number of sequences corresponding to the chromosomal region suspected of having a copy number difference (e.g., at least 1,000 and sometimes at least 5,000 or more sequences) and comparing this value with a similar value for another chromosomal region that is not suspected of having a copy number difference. Being able to count fragments consistently and accurately is paramount to the success of many NIPT methods.

Some NIPT methods use polymerase chain reaction (PCR) to amplify DNA. PCR is widely used, but it suffers from a variety of limitations that may adversely affect the accuracy of the results. PCR can introduce sequence artifacts into the sample and produce amplification bias. PCR sequence artifacts are errors introduced by the PCR reaction into the DNA sequence of the PCR amplified product. PCR sequence artifacts may be caused by a variety of events, such as by forming chimeric molecules (e.g., two different DNA fragments are joined end-to-end), forming heteroduplex DNA (e.g., two different DNA molecules hybridize with each other), and by errors generated by amplification enzymes (e.g., by Taq DNA polymerase setting mismatched nucleotides in the DNA template). Sequence bias from PCR is the skewed distribution of PCR products compared to the original sample. PCR sequence bias can be caused by various events, such as inherent differences in template amplification efficiency or inhibition of amplification due to self-annealing of the DNA template. PCR errors result in unequal amplification of different DNA molecules, so that the amplified sample no longer represents the original sample. PCR is also known to be sensitive to exogenous DNA contamination from the environment. Due to the exponential amplification of DNA during PCR, even very small amounts of exogenous DNA contamination in PCR reactions can lead to highly inaccurate results. Exogenous DNA contamination can be introduced from aerosolized droplets in the air or can be transferred into the reaction from contaminated equipment.

Using rolling circle amplification (RCA) to analyze cfDNA from maternal blood avoids many of the problems associated with PCR. However, RCA products are not easily quantified in a statistically robust manner. On a practical level, while the absolute number of products in an RCA reaction may be high enough to provide statistical robustness, different RCA products can amplify and detect at varying efficiencies. Therefore, consistently detecting tens or hundreds of thousands of RCA products has been challenging.

Overview

In conjunction with other, the probe system of analyzing nucleic acid sample is described herein.Probe can be designed in such a way that they can be connected to the target fragment (herein also referred to as " target sequence " or only " fragment ") of the genomic DNA from different loci (for example, different chromosomes) to produce circular DNA molecules.Circular DNA molecules, even if they contain the fragment from different chromosomes, all contain identical " backbone " sequence.In addition, in some embodiments, all circular DNA molecules containing the fragment from the same locus all contain identical locus specific marker sequence (identifier sequence), that is, locus specific barcode.In these embodiments, the primer amplification circular DNA molecule with sequence hybridization in the main chain can be used, and the oligonucleotide hybridization of labeling can be carried out by RCA product, and the locus of derived cloned fragment can be detected, wherein the oligonucleotide of labeling hybridizes with locus specific marker sequence.As will be apparent, multiple locus specific marker sequences and the oligonucleotide of labeling that can be distinguished with these sequence hybridizations can be used, this embodiment of the method of multiplexing. Because all circular products have the same backbone and differ from each other only by the sequence of the cloned fragment and the locus-specific barcode, RCA products amplified from these products are consistently amplified, and the loci corresponding to these RCA products can be accurately detected. Methods utilizing this probe system and kits for performing the methods are also provided.

As will be discussed in more detail below, in certain cases, the method can be used to detect chromosomal abnormalities (e.g., trisomy 21) in the fetus using a sample of cfDNA from a pregnant woman carrying the fetus.

A probe system for analyzing a nucleic acid sample is provided. In some embodiments, the probe system can comprise: (a) a set of marker oligonucleotides of sequence B; (b) a set of splint oligonucleotides of the formula X'-A'-B'-Z', wherein: in the set: (i) sequences A' and B' vary, and (ii) sequences X' and Z' are different from each other and are not variable; and, in each splint oligonucleotide: (i) sequence A' is complementary to a genomic fragment of the nucleic acid sample and (ii) sequence B' is complementary to at least one member of the set of marker oligonucleotides; and (c) one or more probe sequences comprising X and Z, wherein sequences X and Z are not variable and hybridize to sequences X' and Z'; wherein each splint oligonucleotide is capable of hybridizing to: (i) the probe sequence, (ii) a member of the set of marker oligonucleotides, and (iii) a genomic fragment, thereby generating a ligatable complex of the formula X-A-B-Z. In some embodiments, different marker oligonucleotides and their complementary sequence B' identify different chromosomes, for example, chromosomes 21, 18, and 13.

In some embodiments, the collection of marker oligonucleotides can comprise at least two (e.g., 2, 3, or 4 or more) different B-sequence marker oligonucleotides, and there are at least 100 different A' sequences and at least two different B' sequences complementary to the at least two different marker oligonucleotides in the collection of splint oligonucleotides.

In some embodiments, each marker oligonucleotide or its complementary B' sequence in a splint oligonucleotide can correspond to a genomic fragment.

In some embodiments, each marker oligonucleotide or its complementary B' sequence in a splint oligonucleotide can indicate the locus in the genome from which the genomic fragment originates.

In some embodiments, each marker oligonucleotide or its complementary B' sequence in a splint oligonucleotide can indicate the chromosome from which the genomic fragment originated.

In some embodiments, the genomic fragment is from a mammalian genome.

In some embodiments, each marker oligonucleotide or its complementary B' sequence in the splint oligonucleotide can identify one or more of chromosome 21, chromosome 18, and chromosome 13.

In some embodiments, the genomic fragments may be restriction fragments.

In some embodiments, the one or more probe sequences of (c) further comprise an oligonucleotide comprising sequence Y, and wherein the ligatable complex is linear.

In some embodiments, the probe system can also be a PCR primer pair that hybridizes to one or more probes of (c).

In some embodiments, one or more probe sequences of (c) can comprise a backbone probe of the formula X-Y-Z, wherein Y comprises an oligonucleotide sequence, such that the ligatable complex is a circular ligatable complex of the formula X-A-B-Z-Y, wherein sequence Y joins sequences X and Z.

In some embodiments, the probe system can further comprise a rolling circle amplification primer that hybridizes to a sequence in the backbone probe.

In some embodiments, the probe system can further comprise (A) a rolling circle amplification primer that hybridizes a sequence to the backbone probe; and (B) up to four distinguishably labeled detection oligonucleotides, wherein each distinguishably labeled detection oligonucleotide hybridizes to the B' sequence.

Also provided is a method for analyzing a sample. In some embodiments, the method can include: (a) hybridizing any embodiment of the probe system summarized above to a test genomic sample comprising genomic fragments to produce a ligatable complex of the formula X-A-B-Z; (b) ligating the ligatable complex to produce product DNA molecules of the formula X-A-B-Z; and (c) counting the product DNA molecules corresponding to each locus marker of sequence B.

In some embodiments, counting can be performed by sequencing the product DNA molecules or amplified products thereof to generate sequence reads, and counting the number of sequence reads comprising each sequence B or its complement.

In some embodiments, the product DNA molecules may be circular, and counting may comprise amplifying the product DNA molecules by rolling circle amplification and counting the number of amplification products comprising each sequence B or its complement. In these embodiments, the method may comprise labeling the RCA products using a distinguishably labeled probe that hybridizes to sequence B', and performing the counting by counting the number of RCA products for each distinguishable label.

In some embodiments, the method may comprise: i. depositing the RCA products on a planar support; and ii. counting the number of individually labeled RCA products in a region of the support. In these embodiments, the support may be, for example, a glass slide or a porous transparent capillary membrane.

In some embodiments, different sequences B and their complementary sequences B' identify different chromosomes, and the method further comprises comparing the number of product DNA molecules comprising the first sequence of B or B' with the number of product DNA molecules comprising the second sequence of B or B' to determine whether the genomic sample has aneuploidy.

In some embodiments, the method can include comparing the count results of step (c) to count results obtained from one or more reference samples.

In some embodiments, the test genomic sample can be from a patient suspected of having or at risk of having a disease or condition, and the counting result of step (c) provides an indication of whether the patient or their fetus has the disease or condition.

In some embodiments, the disease or condition may be cancer, an infectious disease, an inflammatory disease, transplant rejection, or a trisomy.

In some embodiments, the fragments are restriction fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled person will understand that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

FIG1 schematically illustrates some features of the probe system of the present invention.

FIG2 schematically shows how sequence B serves to identify the locus of sequence A.

FIG3 schematically shows some exemplary probe system layouts.

FIG4 schematically illustrates some features of an embodiment of the subject method.

FIG5 schematically illustrates some features of one embodiment of the subject method.

Figure 6 schematically shows the design of the probe system.

Figure 7 shows data obtained using two different probe systems.

Figure 8 shows data obtained from analysis of clinical samples.

definition

Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the specification.

Numerical ranges are inclusive of the numbers defining the range.Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxyl orientation, respectively.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., John Wiley and Sons, New York (1994) and Hale and Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide those of skill in the art with a general meaning of many of the terms used herein. In addition, for clarity and ease of reference, certain terms are defined below.

It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. For example, the term "primer" refers to one or more primers, i.e., a single primer and multiple primers. It is further noted that these claims can be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for the use of such exclusive terminology as "solely," "only," and the like in connection with describing claim elements, or the use of a "negative" limitation.

The term "nucleotide" is intended to include those moieties that contain not only the known purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the term "nucleotide" includes moieties that contain haptens or fluorescent markers and may contain not only conventional ribose and deoxyribose sugars but also other sugars. Modified nucleosides or nucleotides also include modifications on the sugar moiety, for example, where one or more hydroxyl groups are replaced with halogen atoms or aliphatic groups, functionalized into ethers, amines, etc.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe polymers of any length (e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases) composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and that can be produced enzymatically or synthetically (e.g., PNAs as described in U.S. Pat. No. 5,948,902 and references cited therein) that can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. Naturally occurring nucleotides include guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively). DNA and RNA have deoxyribose and ribose sugar backbones, respectively, while the backbone of PNAs is composed of repeating N-(2-aminomethyl)-glycine units linked by peptide bonds. In PNA, various purine and pyrimidine bases are linked to the backbone via methylene carbonyl bonds. Locked nucleic acids (LNA), often referred to as inaccessible RNA, are modified RNA nucleotides. The ribose moiety of an LNA nucleotide is modified with an additional bridge connecting the 2' oxygen and the 4' carbon. This bridge "locks" the ribose sugar in a 3'-endo (North) conformation, which is often found in A-form duplexes. Whenever desired, LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides. The term "unstructured nucleic acid" or "UNA" refers to a nucleic acid containing non-natural nucleotides that are bound to each other with reduced stability. For example, an unstructured nucleic acid can contain G' and C' residues, where these residues correspond to non-naturally occurring forms of G and C, i.e., analogs, that base pair with each other with reduced stability but retain the ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acids are described in US 20050233340, which is incorporated herein by reference for its UNA disclosure.

As used herein, term " oligonucleotide " refers to about 2 to 200 nucleotides, up to a nucleotide single-stranded polymer of 500 nucleotides in length. Oligonucleotide can be synthetic or can be enzymatically produced, and in some embodiments has a 30 to 150 nucleotide length. Oligonucleotide can contain ribonucleotide monomers (that is, can be oligoribonucleotides) or deoxyribonucleotide monomers. Oligonucleotide can for example have 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotide length.

As used herein, the term "primer" refers to an oligonucleotide that can serve as a starting point for synthesis when placed under conditions that cause the synthesis of a primer extension product complementary to a nucleic acid chain (i.e., in the presence of nucleotides and an inducing substance (such as DNA polymerase) and at a suitable temperature and pH). The primer can be single-stranded and must be long enough to initiate the synthesis of the desired extension product in the presence of an inducing substance. The exact length of the primer will depend on numerous factors, including the temperature, the source of the primer, and the purposes of the method. For example, for diagnostic applications, depending on the complexity of the target sequence or fragment, an oligonucleotide primer generally contains 15-25 or more nucleotides, but it can contain fewer nucleotides. Primers herein are selected to be substantially complementary to the different chains of a specific target DNA sequence. This means that primers must be fully complementary to hybridize with their corresponding chains. Therefore, the primer sequence does not need to reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be connected to the 5' end of the primer, and the remainder of the primer sequence is complementary to the chain. Alternatively, non-complementary bases or longer sequences may be interspersed into the primer, provided that the primer sequence is sufficiently complementary to the sequence of the strand to hybridize therewith, thereby forming a template for the synthesis of extension products.

The term "hybridization" refers to a process in which a nucleic acid chain anneals with a second complementary nucleic acid chain under normal hybridization conditions and forms a stable duplex (homoduplex or heteroduplex) and does not form a stable duplex with an unrelated nucleic acid molecule under identical normal hybridization conditions. The formation of a duplex is completed by annealing two complementary nucleic acid chains in a hybridization reaction. Hybridization reaction can be made highly specific in the following manner: the hybridization conditions (often referred to as hybridization stringency) in which the hybridization reaction occurs are adjusted, so that the hybridization between the two nucleic acid chains will not form a stable duplex, for example, the duplex in the double-stranded region is retained under normal stringency conditions, unless the two nucleic acid chains contain a certain number of nucleotides in a specific sequence that is substantially or completely complementary. "Normal hybridization or normal stringency conditions" of any given hybridization reaction is easily determined. See, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. As used herein, the term "hybridization" refers to any process by which a strand of nucleic acid molecules binds to a complementary strand through base pairing.

A nucleic acid is considered "selectively hybridizable to" a reference nucleic acid sequence if it specifically hybridizes to the reference nucleic acid sequence under high stringency hybridization and wash conditions. Moderate and high stringency hybridization conditions are known (see, e.g., Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001 Cold Spring Harbor, N.Y.). An example of high stringency conditions includes hybridization in 50% formamide, 5x SSC, 5x Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured carrier DNA at about 42°C, followed by two washes in 2x SSC and 0.5% SDS at room temperature and two additional washes in 0.1x SSC and 0.5% SDS at 42°C.

As used herein, the term "barcode sequence" or "molecular barcode" refers to a unique nucleotide sequence used to a) identify and/or trace the source of a polynucleotide in a reaction and/or b) count how many times an initial molecule has been sequenced (e.g., in the case where substantially every molecule in a sample is tagged with a different sequence and the sample is subsequently amplified). The barcode sequence can be at the 5' end, 3' end, or in the middle of an oligonucleotide. Barcode sequences can vary widely in size and composition; the following references provide guidance for selecting a barcode sequence set suitable for a particular embodiment: Casbon (Nuc. Acids Res. 2011, 22e81); Brenner U.S. Pat. No. 5,635,400; Brenner et al., Proc. Natl. Acad. Sci., 97: 1665-1670 (2000); Shoemaker et al., Nature Genetics, 14: 450-456 (1996); Morris et al., European Patent Publication 0799897A1; Wallace, U.S. Pat. No. 5,981,179, etc. In specific embodiments, the barcode sequence can have a length ranging from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides.

As used herein, the term "sequencing" refers to methods by which the identity of at least 10 consecutive nucleotides of a polynucleotide is obtained (e.g., the identity of at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotides).

The term "next generation sequencing" refers to so-called parallelized synthesis sequencing platforms or ligation sequencing platforms currently used by, for example, Illumina, Life Technologies, and Roche. Next generation sequencing methods may also include nanopore sequencing methods or methods based on electronic detection, such as, for example, the Ion Torrent technology commercialized by Life Technologies.

As used herein, the terms "duplex" or "duplexed" describe two complementary polynucleotides that are base paired (ie, hybridized together).

The terms "determine," "measure," "evaluate," "assess," "verify," and "analyze" are used interchangeably herein to refer to various forms of measurement and include determining whether an element is present or absent. These terms encompass quantitative and/or qualitative determinations. Assessments can be relative or absolute.

As used herein, the term "affinity tag" refers to a portion that can be used to separate molecules connected to the affinity tag from other molecules that do not contain the affinity tag. An "affinity tag" is a member of a specific binding pair (i.e., two molecules in which one molecule specifically binds to the other molecule by chemical or physical means). The complementary member of the specific binding pair (referred to herein as a "capture agent") can be immobilized (e.g., to a chromatography support, a bead, or a planar surface) to produce an affinity chromatography support that specifically binds to the affinity tag. In other words, an "affinity tag" can be combined with a "capture agent," wherein the affinity tag specifically binds to the capture agent, thereby facilitating the separation of molecules connected to the affinity tag from other molecules that do not contain the affinity tag.

As used herein, the term "biotin moiety" refers to an affinity agent comprising biotin or a biotin analog, such as desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin, and the like. The biotin moiety binds to streptavidin with an affinity of at least 10^{^-8} M. The biotin affinity agent may further comprise a linker, for example, ─LC-biotin, ─LC-LC-biotin, ─SLC-biotin, or ─PEG_{_n}-biotin, where n is 3-12.

As used herein, the term "terminal nucleotide" refers to the nucleotide at the 5' end or the 3' end of a nucleic acid molecule. Nucleic acid molecules can be in double-stranded form (i.e., duplex) or in single-stranded form.

As used herein, the term "ligation" refers to the enzymatically catalyzed joining of the terminal nucleotide at the 5' end of a first DNA molecule to the terminal nucleotide at the 3' end of a second DNA molecule.

The terms "plurality,""collection," and "population" are used interchangeably to refer to an entity comprising at least 2 members. In some cases, a plurality can have at least 10, at least 100, at least 100, at least 10,000, at least 10,000, at least 10 ⁶ , at least 10 ⁷ , at least 10 ⁸ , or at least 10 ⁹ or more members.

The term "digestion" is intended to indicate the process by which a nucleic acid is cleaved by a restriction enzyme. To digest a nucleic acid, a restriction enzyme and a nucleic acid containing a recognition site for the restriction enzyme are contacted under conditions suitable for the activity of the restriction enzyme. Conditions suitable for the activity of commercially available restriction enzymes are known and are provided with these enzymes when purchased.

An "oligonucleotide binding site" refers to a site in a target polynucleotide or fragment to which an oligonucleotide hybridizes. If an oligonucleotide "provides" a binding site for a primer, then the primer can hybridize to the oligonucleotide or its complement.

As used herein, the term "separate" refers to the physical separation of two elements (eg, by size or affinity, etc.) as well as the degradation of one element, leaving the other element intact.

As used herein, the term "reference chromosomal region" refers to a chromosomal region whose nucleotide sequence is known, such as a chromosomal region whose sequence is deposited, for example, in the NCBI Genbank database or other databases.

As used herein, the term "strand" refers to a nucleic acid composed of nucleotides covalently linked together by covalent bonds (eg, phosphodiester bonds).

In cell, DNA exists in double-stranded form conventionally, so has two nucleic acid complementary chains, referred to as "top" chain and "bottom" chain in this article.In some cases, the complementary chain of chromosome region can be referred to as "plus" and "minus" chain, "first" chain and "second" chain, "coding" chain and "non-coding" chain, "Watson" chain and "Crick" chain or "righteousness" and "antisense" chain.Chain is attributed to top or bottom chain is arbitrary and does not imply any specific orientation, function or structure.The nucleotide sequence of the first chain of several exemplary mammal chromosome regions (for example, BAC, assembly, chromosome etc.) is known, and can for example be found in the NCBI Genbank database.

As used herein, the term "top strand" refers to either strand of a nucleic acid, not both strands of a nucleic acid. When an oligonucleotide or primer binds or anneals only to the top strand, it binds only to one strand, but not to the other strand. As used herein, the term "bottom strand" refers to the strand that is complementary to the "top strand." When an oligonucleotide binds or anneals only to one strand, it binds only to one strand (e.g., the first strand or the second strand), but not to the other strand.

The term "covalently linking" refers to the creation of a covalent bond between two separate molecules (eg, the top and bottom strands of a double-stranded nucleic acid). Ligating is a type of covalent linking.

As used herein, the term "denaturation" refers to subjecting a duplex to suitable denaturing conditions to separate at least a portion of the base pairs of the nucleic acid duplex. Denaturing conditions are well known in the art. In one embodiment, to denature a nucleic acid duplex, the duplex can be exposed to a temperature at the melting temperature of the duplex, thereby releasing one strand of the duplex from the other. In certain embodiments, the nucleic acid can be denatured by exposing the nucleic acid to a temperature of at least 90° C. for an appropriate amount of time (e.g., at least 30 seconds up to 30 minutes). The nucleic acid can also be denatured chemically (e.g., using urea or NaOH).

As used herein, the term "label" refers to any atom or molecule that can be used to provide a detectable (preferably quantifiable) effect and that can be attached to a nucleic acid or protein. Labels include, but are not limited to, dyes and radiolabels such as ³² P, alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET); binding moieties such as biotin; haptens such as digoxin; activated luminescent, phosphorescent or fluorescent moieties; and fluorescent dyes. Labels can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric analysis, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like. Labels can be charged moieties (positive or negative) or, alternatively, can be electrically neutral. Labels can comprise or consist of a nucleic acid or protein sequence, as long as the sequence comprising the label is detectable.

As used herein, the terms "labeled oligonucleotide" and "labeled probe" refer to oligonucleotides having affinity tags (e.g., biotin moieties), oligonucleotides modified with atoms or groups that allow separation or detection (e.g., bromo-deoxyuridine or colloidal gold particles that impart different densities), and oligonucleotides modified with optically detectable labels (e.g., fluorescent or another type of light-emitting label). Oligonucleotides containing only naturally occurring nucleotides are not labeled oligonucleotides.

As used herein, the term "extension" refers to extending a primer by adding nucleotides using a polymerase. If a primer annealed to a nucleic acid is extended, the nucleic acid serves as a template for the extension reaction.

As used herein, in the phrase "ligating a first and a second oligonucleotide to corresponding ends of a fragment," the term "corresponding ends" means adding one oligonucleotide to one end of the fragment and adding another oligonucleotide to the other end of the target fragment.

As used herein, the term "ligatably adjacent" in the context of two oligonucleotide sequences that are ligatably adjacent to each other means that there are no intervening nucleotides between the two oligonucleotides and they can be ligated to each other.

As used herein, the term "splint oligonucleotide" refers to an oligonucleotide that, when hybridized to two or more other polynucleotides, acts as a "splint" to position these polynucleotides adjacent to each other so that they can be ligated together, as shown in FIG1 .

As used herein, the term "circular nucleic acid molecule" refers to a chain in the form of a closed circle with no free 3' or 5' termini.

As used herein, the term "corresponding to" and grammatical equivalents, such as "corresponding to", refer to a specific relationship between the elements to which the term refers. For example, an RCA corresponding to a sequence in a genome contains the same nucleotide sequence as the sequence in the genome.

Certain polynucleotides described herein may be referred to by a formula (e.g., "X'-A'-B'-Z'"). Unless otherwise indicated, a polynucleotide defined by a formula can be oriented in the 5' to 3' direction or the 5' to 3' direction. For example, a polynucleotide defined by the formula "X'-A'-B'-Z'" can be "5'-X'-A'-B'-Z'-3'" or "3'-X'-A'-B'-Z'-5'." The components of a formula, e.g., "A," "X," and "B," etc., each refer to a separately definable sequence of nucleotides in a polynucleotide, wherein, unless otherwise indicated by the context (e.g., in the context of a "linkable complex of a particular formula"), the sequences are covalently linked together so that the polynucleotide described by a certain formula is a single molecule. In many cases, the components of a formula are immediately adjacent to each other in a single molecule. Following convention, the complement of a sequence shown in a formula will be indicated by a single quote ('), so that the complement of sequence "A" will be "A'." In addition, unless otherwise indicated or indicated by the context, a polynucleotide defined by a certain formula may have additional sequences, primer binding sites, molecules at its 3' terminus, its 5' terminus, or both at its 3' terminus and its 5' terminus. Barcode, promoter or spacer sequence etc. If the polynucleotide limited by a certain formula will be described as circular, the ends of these molecules are directly or indirectly connected together. For example, in the case of a cyclic complex of formula X-A-B-Z-Y, the 5' end of the molecule is directly or indirectly connected to the 3' end of the molecule to produce a ring. As will be apparent, the various component sequences of polynucleotides (e.g., A, B, C, X, Y, Z, etc.) can independently be any desired length, as long as they can perform the desired function (e.g., hybridize with another sequence). For example, the various component sequences of polynucleotides can independently have a length in the range of 8-80 nucleotides (e.g., 10-50 nucleotides or 12-30 nucleotides).

The term "ligatable complex" (e.g., of formula X-A-B-Z) refers to a complex in which multiple oligonucleotides (in circular or linear form) are ligatably adjacent to each other, held together by a splint oligonucleotide, as shown in Figure 1.

The term "ligatable circular complex" (e.g., of the formula X-A-B-Z-Y) refers to a circular complex in which multiple oligonucleotides are ligatably adjacent to each other in a loop, held together by a splint oligonucleotide.

As used herein, the terms "locus" and "genomic locus" refer to a defined region of a genome (e.g., an animal or plant genome such as a human, monkey, rat, fish, insect, or plant genome). A locus can be a chromosomal region as short as 100 kb and can be as long as a chromosome arm or an entire chromosome.

The terms "first locus" and "second locus" refer to different loci, ie, different regions in the genome, eg, different chromosome arms or different chromosomes.

The term "fragment of a locus" refers to a population of defined fragments of a particular locus (which can be generated using restriction enzymes or with the aid of RNA-guided reprogramming endonucleases such as CAS9). Not all fragments of a locus need to be analyzed. Because the sequences of many genomes are already public, it is routine to design oligonucleotides that hybridize to a certain fragment of a locus.

The term "complementary to a fragment" refers to a sequence that is complementary to a strand (top strand or bottom strand) of a fragment.

As used herein, the term "genomic sequence" refers to a sequence present in a genome.

In the context of two or more variable nucleic acid sequences, the term "variable" refers to two or more nucleic acids that have different nucleotide sequences relative to each other. In other words, if a population of polynucleotides has variable sequences or a particular sequence "varies," the nucleotide sequence of the polynucleotide molecules in the population varies from molecule to molecule. The term "variable" should not be interpreted as requiring that each molecule in the population have a sequence that is different from that of other molecules in the population.

If two nucleic acids (e.g., sequences A and A') are "complementary", they hybridize to each other under high stringency conditions. In many cases, two sequences that are complementary have at least 10, e.g., at least 12, at least 15, at least 20, or at least 25 nucleotides of complementarity and in some cases may have one, two, or three non-complementary bases.

In the context of identifying sequences of a locus, the term "identification" refers to a molecular barcode that is unique to the locus. This sequence does not come from the locus itself, but rather is a molecular barcode that is added to the fragments of the locus being analyzed and identifies them as coming from that locus, typically with sequences that are not present in the sample being analyzed. For example, if a fragment from a first locus is linked to a first marker sequence and a fragment from a second locus is linked to a second marker sequence, the source of the fragments (the loci to which they correspond) can be determined by detecting which marker sequence has been linked to the fragments.

The term "reverse" in the context of two sequences hybridizing in reverse direction to the other sequence refers to a structure in which the 5' and 3' ends of one of the sequences hybridize to the other sequence in such a manner that the ends face each other, as shown at the top of Figure 3B.

As used herein, the term "rolling circle amplification" or (abbreviation) "RCA" refers to the isothermal amplification of linear concatenated copies of circular nucleic acid templates using a strand displacement polymerase. RCA is well known in the field of molecular biology and is described in various publications, including but not limited to Lizardi et al. (Nat. Genet. 1998 19: 225-232), Schweitzer et al. (Proc. Natl. Acad. Sci. 2000 97: 10113-10119), Wiltshire et al. (Clin. Chem. 2000 46: 1990-1993) and Schweitzer et al. (Curr. Opin. Biotech 2001 12: 21-27), which are incorporated herein by reference.

As used herein, the term "rolling circle amplification product" refers to the concatemer product of a rolling circle amplification reaction. As used herein, the term "fluorescently labeled rolling circle amplification product" refers to a rolling circle amplification product that has been fluorescently labeled, for example, by hybridization of a fluorescently labeled oligonucleotide to the rolling circle amplification product or other means (e.g., by incorporation of fluorescent nucleotides into the product during amplification).

As used herein, the term "region" in the context of a region of a support or a region of an image refers to a continuous or discontinuous region. For example, if the method involves determining and counting the number of labeled RCA products within a region, the region in which the RCA products are to be counted may be a single continuous space or a plurality of discontinuous spaces.

As used herein, the term "imaging" refers to the process by which optical signals from an object's surface are detected and data associated with the locations (i.e., "pixels") are stored. A digital image of the object can be reconstructed from this data. A single image or one or more images can be used to image an area of a support.

As used herein, the term "individual labelled RCA products" refers to individual RCA molecules that are labelled.

As used herein, the term "counting" refers to determining the number of individual objects in a larger collection. "Counting" requires detecting individual signals from each object in a plurality of objects (not the collective signal from the plurality of objects) and then determining how many objects are present in the plurality of objects by counting the individual signals. In the context of the present method, "counting" is performed by determining the number of individual signals in a signal array.

As used herein, when referring to an array of RCA products, the term "array" refers to a collection of individual RCA products on a planar surface, wherein the RCA products are spatially separated from one another in the plane of the surface (to the extent that the array is truly random according to a Poisson distribution). A "random" array is one in which the elements (e.g., RCA products) are distributed in unpredicted positions on the surface of the substrate. In some cases, the distribution of RCA products in a random array can be described by Poisson statistics, such that, for example, the distribution of distances between RCA products of a random array is approximated by a Poisson distribution.

Other term definitions may appear throughout this specification.

Description of Exemplary Embodiments

Before describing various embodiments, it should be understood that the teachings of the present disclosure are not limited to the specific embodiments described and may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present teachings will be limited only by the appended claims.

The section headings used herein are for organizational purposes only and are not to be construed in any way as limiting the subject matter described. Although the present teachings are described in conjunction with various embodiments, the present teachings are not intended to be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those skilled in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein may also be used for the implementation or testing of the present teachings, some exemplary methods and materials are now described.

The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Furthermore, the dates of publication provided may be different from the actual publication dates which need to be independently verified.

As will be apparent to those skilled in the art upon reading this disclosure, each of the various variations described and illustrated herein has independent components and features that can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the teachings of the present invention. Any of the methods described can be practiced in the order of events recited or in any other order that is logically possible.

All patents and publications mentioned herein, including all sequences disclosed within such patents and publications, are expressly incorporated by reference.

Probe composition

Some embodiments of the probe system can include: (a) a set of marker oligonucleotides of sequence B; (b) a set of splint oligonucleotides of the formula X'-A'-B'-Z', wherein: within the set: (i) sequences A and B' vary, and (ii) sequences X' and Z' are different from each other and are not variable; and, in each splint oligonucleotide: (i) sequence A' is complementary to a genomic fragment of a nucleic acid sample and (ii) sequence B' is complementary to at least one member of the set of marker oligonucleotides; and (c) one or more probe sequences comprising X and Z, wherein sequences X and Z are not variable and hybridize to sequences X' and Z'; wherein each splint oligonucleotide is capable of hybridizing to: (i) the probe sequence, (ii) a member of the set of marker oligonucleotides, and (iii) a genomic fragment, thereby generating a ligatable complex of the formula X-A-B-Z. As will be described in more detail below, in some embodiments, different marker oligonucleotides and their complementary sequence B' identify different chromosomes, for example, chromosomes 21, 18, and 13.

Fig. 1 shows the connectable complex of formula XABZ, and its structural characterization probe system of the present invention.As shown in Figure 1, in complex, sequence X, A, B and Z are adjacent to each other in connectable ground, are fixed in place by splint oligonucleotide.As shown in Figure 1, sequence A is genomic target fragment (for example, the chain of restriction fragment), and sequence B identifies the locus (for example, specific region on chromosome, specific chromosome arm or specific chromosome etc.) of derived adjacent sequence A.The relationship between sequence A and B is shown in 2 figures, and described Fig. 2 shows the simple probe set of hybridization with various genomic fragments (A ₁ to A ₆ ).As shown in Figure 2, the genomic fragment in the top three complexes (sequence A ₁ , A ₂ and A ₃ ) is from the first locus (for example, chromosome 21) and the genomic fragment in the bottom three complexes (sequence A ₄ , A ₅ and A ₆ ) is from the second locus (for example, chromosome 18). The loci from which the genomic fragments in the top three complexes were derived are identified by a single sequence (B ₁ ), and the loci from which the genomic fragments in the bottom three complexes were derived are identified by different sequences (B ₂ ). Sequences X and Z are identical in all shown complexes.

As will be apparent, the splint oligonucleotide set can be as complex as desired, and in some embodiments, sequence A' can have a complexity of at least 100, at least 1,000, at least 5,000, at least 10,000, or at least 50,000, or more, meaning that the splint oligonucleotide can, in general, hybridize to at least 100, at least 1,000, at least 5,000, at least 10,000, or at least 50,000, or more genomic DNA fragments. Sequence B' in the splint oligonucleotide set can have much less diversity because it simply acts as a locus marker. Thus, in the splint oligonucleotide set, sequence B' can have a complexity of at least 2 (e.g., 3 or 4), although in some embodiments, sequence B' can have a complexity of at least 10, at least 100, or at least 1000. As will be apparent, because sequence B' is complementary to sequence B, the complexity of the locus-specific oligonucleotide set can be the same as the complexity of sequence B'. For example, if there are three marker oligonucleotides, there can be three different B' sequences. The number of splint oligonucleotides in a collection can vary widely, depending on the length of the locus and the number of target fragments. In some embodiments, each splint oligonucleotide collection can contain at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, or at least 50,000 different splint oligonucleotides.

For example, in some embodiments, a collection of splint oligonucleotides can contain: (i) a first subpopulation of splint oligonucleotides containing at least 100 A' sequences (e.g., set A1 _,X ', x=1-100+) that are complementary to different fragments of a first locus (e.g., fragments of chromosome 21, or, for example, set _A1,X , x=1-100+), wherein each of this subpopulation of splint oligonucleotides has the same B' sequence, e.g., _B1 '; (ii) a second subpopulation of splint oligonucleotides containing at least 100 A' sequences (e.g., set A2 _,X ', x=1-100+) that are complementary to different fragments of a second locus (e.g., fragments of chromosome 18, or, for example, set _A2X , x=1-100+), wherein each of this subpopulation of splint oligonucleotides has the same B' sequence that is different from the B' sequence of the first (or any other) subpopulation, e.g., _B2 '; (iii) a third subpopulation of splint oligonucleotides containing at least 100 A' sequences (e.g., set A3 _,X ', x=1-100+) that are complementary to different fragments of a third locus (e.g., fragments of chromosome 18, or, for example, set _A3X , x=1-100+), wherein each of this subpopulation of splint oligonucleotides has the same B' sequence that is different from the B' sequences of any other subpopulation, e.g., _B3 '; (iv) an optional fourth subpopulation of splint oligonucleotides containing at least 100 A' sequences (e.g., set _A4X ', x=1-100+) that are complementary to different fragments of a fourth locus (e.g., fragments of another chromosome or, for example, set A4 _,X , x=1-100+), wherein each of this subpopulation of splint oligonucleotides has a B' sequence that is different from the B' sequences of any other subpopulation, e.g., B4 _' .

As shown in Figure 3, the probe system can be arranged in a variety of different ways, depending on how it will be used. For example, as shown in Figure 3A, Figure 3C and Figure 3D, sequences X and Z can be in different molecules, and therefore, the connectable complex is linear. In these embodiments, one or more probes containing sequences X and Y can include a first oligonucleotide comprising sequence X and a second oligonucleotide comprising sequence Y. In these embodiments, the first and second oligonucleotides do not need to be tailed, as shown in Figure 1A. In these embodiments, after connection, the connection product can be amplified, using, for example, a tailed (talked) PCR primer amplification hybridized with sequences X and Z. In some embodiments (as shown in Figure 3C and Figure 3D), the first and/or second oligonucleotide can itself have a tail that provides a primer binding site to promote amplification and counting. In some embodiments, the tail can contain a molecular indexer (for example, a random sequence), which allows counting the number of original connection products after those molecules have been amplified and sequenced. In optional embodiments and as shown in Figure 3B, the one or more probes containing sequences X and Y can be a single backbone probe of formula X-Y-Z. In these embodiments and as shown, the ligatable complex is a circular ligatable complex of the formula X-A-B-Z-Y, wherein sequence Y joins sequences X and Z. In another embodiment, shown in Figure 3E , one or more probes containing sequences X and Z can themselves be part of a splint oligonucleotide. In these embodiments, the ligation product can be a "dumbbell" shape, as shown in Figure 3E .

In these embodiments, the probe system can also include a PCR primer pair that hybridizes to one or more probes comprising sequences X and Z, thereby allowing amplification of the central portion of the connection product (i.e., the portion containing sequences A and B). In some embodiments, such as the embodiment shown in Figure 3B, the probe system can also include a rolling circle amplification primer that hybridizes to the sequence in the backbone probe, thereby promoting the amplification of these products by rolling circle amplification. In some embodiments, the probe system can include a rolling circle amplification primer that hybridizes the sequence to the backbone probe; and up to four distinguishably labeled oligonucleotides, each of which distinguishably labeled oligonucleotides hybridizes to the complement of sequence B '. This will be explained in more detail below.

Thus, some embodiments of the probe system can include a splint oligonucleotide, a backbone probe, and one or more locus-specific oligonucleotides. The probe system can also include one or more amplification primers, such as a rolling circle amplification primer that hybridizes to a sequence in the backbone probe or a PCR primer pair that hybridizes to a site in the backbone probe, and, optionally, one or more label probes that hybridize to the complement of the locus-specific oligonucleotide.

As shown above, sequence A' varies between different members of the set, and each of A's sequences is designed to complement a different target fragment of the genome. The sequence of A' can vary in length and sequence independently, and in some cases, can be in the range of 8 to 80 nucleotides, for example, 10 to 60 nucleotides in length, depending on the length and sequence of the target fragment. Sequence B' identifies the locus from which the adjacent fragment originates (e.g., a specific chromosome, such as chromosome 18 or 21, etc.). Sequence B' can have any suitable length, but in some embodiments, it is in the range of 8 to 30 nucleotides in length. In any single assay, sequences X' and Z' are different from each other and are not variable. Sequences X' and Z' can have any suitable length, but in some embodiments, they are independently in the range of 8 to 30 nucleotides in length, although longer or shorter sequences can be used. The overall length of the splint oligonucleotide can be in the range of 50 to 200 nucleotides. In some embodiments, the splint oligonucleotide can be biotinylated, thereby allowing the ligation product (discussed below) to be separated from other unligated products before amplification. As will be apparent, sequences X and Z (which can be of any suitable length, but in some embodiments, are independently within the range of 8 to 30 nucleotides in length) are not variable and hybridize to sequences X' and Z'. The locus-specific oligonucleotide has sequence B, which again can be of any suitable length, for example, within the range of 8 to 30 nucleotides in length.

As indicated above, the complexes generated using the above probe system can be linear or circular (as shown in Figure 3). Figure 4 shows some features of the circular embodiment shown in Figure 3B.

As shown in Figure 4, in some embodiments, the probe system can include a set of splint oligonucleotides 2 (formula X'-A'-B'-Z', which can be in the 5' to 3' or 3' to 5' direction), a backbone probe 6 of the formula X-Y-Z, wherein sequences X and Z are not variable and hybridize to sequences X' and Z' in reverse orientation (i.e., so that the ends of the backbone point to each other, as shown), and a set of locus-specific oligonucleotides 8 having sequence B. Sequence Y in the backbone probe can be any convenient length, for example, 20 to 100 nucleotides. The total length of the backbone probe 6 can be in the range of 50 to 300 nucleotides in length, or in some cases longer.

As shown in FIG4 , a probe set is characterized in a plurality of oligonucleotides that can be hybridized with genomic fragments to produce a first set of ligatable circular complexes 10 (i.e., complexes in which the ends of the backbone probes 6, the locus-specific oligonucleotides 8, and the genomic fragments 4 are ligatably adjacent to each other and are secured ligatably adjacent to each other by the splint oligonucleotides 2). As shown in the illustrated example, the backbone probes 6, the locus-specific oligonucleotides 8, and the fragments 4 hybridize with the first splint oligonucleotide 2 to produce a set of ligatable circular complexes 10 of the formula X-A-B-Z-Y, where sequence Y joins sequences X and Z. The fragments 4 present in this set of ligatable circular complexes 10 can be from at least 2, at least 5, at least 10, or at least 50 or more different loci (e.g., different chromosomes), and the identity of the loci (e.g., specific chromosomes) from which the adjacent fragments originate is provided by the locus-specific oligonucleotides 8, which have the same sequence for each locus. In this example, sequences A and A' (sequences corresponding to different genomic fragments) vary, B and B' (locus markers) vary, and sequences X, Y, and Z do not vary.

As will be described in more detail below, in this embodiment, a probe system (which comprises a first set of splint oligonucleotides 2, a backbone probe 6, and a locus-specific oligonucleotide 8) can be hybridized with a sample comprising a fragment of a genome 4 to produce a first set of ligatable circular complexes 10 of the formula X-A-B-Z-Y, as shown. After ligating the ligatable circular complexes to produce a first set of circular DNA molecules 12 of the formula X-A-B-Z-Y, the first set of circular DNA molecules can be amplified by rolling circle amplification (RCA) to produce a first set of RCA products 16. RCA can be performed using rolling circle amplification primers 14 (as shown in FIG4 ) that hybridize to sequences in the backbone probes 6 or PCR primers that hybridize to sites flanking the ligated fragments. Thus, in certain embodiments, the probe system can additionally comprise rolling circle amplification primers 14 (the primers hybridize to sequences in the backbone probes 6) or PCR primer pairs that hybridize to sites flanking the ligated fragments. After RCA, the "origin" of the cloned fragment in a particular RCA product 16 (i.e., the locus from which the cloned genomic fragment originated, for example, a particular chromosome) can then be determined by hybridization of the first labeled oligonucleotide 18 to the complement of sequence B (i.e., B') or by sequencing. As will be apparent, the labeled oligonucleotide 18 may comprise at least some of sequence B. Thus, in certain embodiments, the probe system may further comprise a labeled oligonucleotide that hybridizes to the complement of the first locus-specific oligonucleotide 8.

As will be apparent, if sequences from two or more different loci are to be detected in the same reaction, the probe system may comprise additional distinguishably labelled oligonucleotides, one for each locus marker B, so that two sets of RCA products can be identified simultaneously. In these embodiments, the probe system may also comprise up to four distinguishably labelled oligonucleotides (e.g., _B1 , _B2 , _B3 , _B4 ), wherein each distinguishably labelled oligonucleotide hybridises to the complement of sequence B' (e.g., _B1 ', _B2 ', _B3 ', _B4 ').

As will be apparent, the fragments that hybridize to the splint oligonucleotides are restriction fragments of the genome being analyzed. Additionally, any of the probes, oligonucleotides, or primers described above (e.g., backbone probes) can contain a molecular barcode (e.g., an index sequence such as a random or semi-random sequence) so that each circular DNA molecule can be distinguished based on the combination of cloned fragments and barcodes, thereby allowing counting of how many initial molecules were sequenced, even after the molecules have been amplified (see, e.g., Casbon et al.).

method

Also provided herein is a method comprising: (a) hybridizing a probe system as described above to a test genomic sample comprising a fragment of a genome to produce a ligatable complex of the formula X-A-B-Z; (b) ligating the ligatable complex to produce product DNA molecules of the formula X-A-B-Z; and (c) counting the product DNA molecules corresponding to each locus marker of sequence B. In some embodiments, counting can be performed by sequencing the product DNA molecules or amplified products thereof to produce sequence reads, and counting the number of sequence reads comprising each sequence B.

In the embodiment in which the product DNA molecule is circular, counting can include amplifying the product DNA molecule by rolling circle amplification, and counting the number of amplified products comprising each sequence B. In these embodiments, the method can include using a distinguishably labeled probe labeling RCA product that hybridizes with sequence B, and by counting the number of each distinguishable marker RCA product, counting. The general principle of an implementation of this method is shown in Fig. 4. As will be apparent, the fragment hybridized with the splint oligonucleotide can be (independently) the top chain or bottom chain restriction fragment of the genome being analyzed. These fragments can be produced by digesting the genome with one or more restriction enzymes (for example, a combination of enzymes with four base recognition sequences) and subsequently denaturing the digested sample. Thus, the fragment being cloned has a definite end, thereby allowing the design of splint oligonucleotide to clone these fragments. There are other ways (for example, methods using flap endonucleases, exonucleases, gap fillings, etc.) to produce a fragment with a definite end.

As indicated above, this approach can be multiplexed to provide a means of analyzing two or more different loci, as shown in Figure 5. Referring to Figure 5, a sample containing fragments of genomic DNA 40 can be: a) hybridized with a probe system 42 comprising: (i) a first set of splint probes as described above; (ii) a first locus-specific oligonucleotide as described above; (iii) a second set of splint probes as described above; (iv) a second locus-specific oligonucleotide as described above; and, (v) a backbone probe as described above, to produce a mixture 44 comprising a first set of ligatable circular complexes of the formula X-A-B-Z-Y (the ligatable circular complexes containing fragments from a first locus (e.g., a first chromosome) and fragments from a second locus (e.g., a second chromosome)). Next, the method comprises (b) ligating the ligatable circular complexes to produce a mixture of circular DNA molecules 46 (the mixture comprising the first and second populations of circular DNA molecules), and after treating the sample with an exonuclease to remove linear nucleic acid molecules, (c) amplifying the circular DNA molecules 46 by rolling circle amplification using a single primer that hybridizes to the backbone probe to produce RCA products 48. The locus from which each fragment contained in each RCA product originates can then be identified by hybridizing the RCA products with distinguishably labeled first and second oligonucleotide probes that hybridize to the complement of the locus-specific oligonucleotide present in each product to produce a labeled sample 50. In these embodiments, the method may comprise: (d) respectively: (i) detecting RCA products containing fragments from the first locus using a labeled probe that hybridizes to the first locus marker sequence and (ii) detecting RCA products containing fragments from the second locus using a labeled probe that hybridizes to the second locus marker sequence, wherein the labeled probes are distinguishably labeled. As shown above, after ligation, if the splint oligonucleotide is biotinylated, the circular product can be separated from the unligated product using, for example, streptavidin beads. In either case, the ligated sample can be treated with an exonuclease to remove the linear DNA molecules from the reaction. This principle can be extended to counting the number of ligation products generated for any number of loci (e.g., 3, 4, up to 10 or up to 100 or more loci).

In some embodiments, the detecting step may (d) comprise: (i) depositing the RCA products on a support; and (ii) counting the number of each labeled RCA product labeled with one label and the number of each labeled RCA product labeled with another label in an area of the support. As will be appreciated, hybridization of the labeled oligonucleotides may be performed before or after the RCA products are distributed on the support.

That is, the number of rolling circle amplification products corresponding to each locus can be estimated, for example, by distributing the RCA products on the surface of a support (slide or porous membrane), hybridizing the RCA products using labeled oligonucleotides (e.g., fluorescently labeled oligonucleotides) and then, for example, using a fluorescence reader, counting the number of discrete signals in the region of the support. Labeling can be performed before or after the product has been distributed on the support, and, because each RCA product contains thousands of copies of the same sequence, there should be thousands of binding sites for the labeled oligonucleotides, thereby increasing the signal. In a multiplex embodiment (e.g., wherein the RCA products corresponding to two different loci are counted), the RCA product corresponding to one locus can be labeled with one fluorophore and the RCA product corresponding to the other locus can be labeled with a different fluorophore, thereby allowing different RCA products to be counted separately.

In certain embodiments, the method comprises: (a) filtering a liquid sample containing rolling circle amplification (RCA) products through a porous transparent capillary membrane, thereby concentrating the RCA products on the membrane and producing an array of RCA products; (b) fluorescently labeling the RCA products before or after step (a); and, (c) counting the number of each labeled RCA product in the membrane area, thereby providing an assessment of the number of labeled RCA products in the sample. In some embodiments, the porous transparent capillary membrane can be a porous anodic aluminum oxide membrane. In these embodiments, step (b) can be performed by hybridizing a fluorescently labeled oligonucleotide to the RCA product before or after step (a). In certain embodiments, the method can include imaging the membrane area to produce one or more images and counting the number of each labeled RCA product in the one or more images. Examples of such methods are described in PCT/IB2016/052495, filed May 2, 2016, which is incorporated herein by reference.

Quantifying the signal from each RCA product is meaningful because in many applications (e.g., non-invasive prenatal diagnosis based on cfDNA analysis), the number of fragments corresponding to a specific chromosome (e.g., chromosome 21) needs to be determined quite accurately and without bias. Common analytical methods use PCR, which, as is well known, is a very biased method because some sequences amplify much more efficiently than others. For many diagnostic tasks, this makes PCR-based strategies impractical.

In particular embodiments, a sample may contain multiple RCA product populations (e.g., 2, 3 or 4 or more RCA product populations, such as a first labeled RCA product population and a second RCA product population), wherein the different RCA product populations are distinguishably labeled, meaning that individual members of each RCA product population label can be independently detected and counted, even when the populations are mixed. Suitable distinguishable fluorescent label pairs that can be used in the subject method include, for example, Cy-3 and Cy-5 (Amersham Inc., Piscataway, NJ), Quasar 570 and Quasar 670 (Biosearch Technology, Novato CA), Alexafluor 555 and Alexafluor 647 (Molecular Probes, Eugene, OR), BODIPY V-1002 and BODIPY V1005 (Molecular Probes, Eugene, OR), POPO-3 and TOTO-3 (Molecular Probes, Eugene, OR), and POPRO3 and TOPRO3 (Molecular Probes, Eugene, OR). Other suitable distinguishable detectable labels can be found, for example, in Kricka et al. (Ann Clin Biochem. 39: 114-29, 2002). For example, the RCA product can be labeled with any combination of ATTO, ALEXA, CY, or dimeric cyanine dyes such as YOYO, TOTO, and the like. Other markers may also be used.

In some cases, RCA product populations can be distinguishably marked by marking it with multiple markers, thereby increasing the possibility of multiplexing. For example, in some cases, a population can be marked with two distinguishable dyes (for example, Cy3 and Cy5), and when read, the population will be distinguishable from a population marked with a single dye (for example, Cy3 or Cy5). In some embodiments, the first RCA product population represents a "test" population of the RCA product of the label and the second RCA product population represents an RCA product "reference" population to which the number of the first RCA product can be compared. For example, in some embodiments, the first RCA product population can correspond to a first chromosome region (for example, a first chromosome such as chromosome 21) and the second RCA product population can correspond to a second chromosome region (for example, a second chromosome such as chromosome 13 or 18 or a different region of the first chromosome), and the number of the first RCA product population and the second RCA product population can be counted and compared to determine whether there is a copy number difference in the region (prompting the presence of duplication or deletion in the test region). In some embodiments, the sample contains at least a first RCA product population and a second RCA product population, wherein the first and second labeled RCA product populations are distinguishably labeled in the labeling step (step (b)). In these embodiments, the method includes counting the number of first labeled RCA products in a membrane area and counting the number of second labeled RCA products in a membrane area (the same area or a different area), thereby providing an assessment of the quantity of the first and second RCA product populations in the sample. This embodiment may also involve comparing the number of the first RCA product in the sample with the number of the second RCA product in the sample.

In certain of these embodiments of the method, the method may comprise imaging the first and second labeled RCA product populations to produce one or more images (e.g., a first image and a second image, respectively) and, optionally, (i) counting the number of labeled RCA products in the one or more images, thereby providing an assessment of the number of first and second labeled RCA product populations in the sample. The first and second labeled RCA product populations may be detected separately using known methods (e.g., using suitable filters, etc.). These embodiments of the method may further comprise comparing the number of first labeled RCA products in the sample with the number of second labeled RCA products in the sample. This step of the method may involve counting at least 1,000 (e.g., at least 5,000, at least 10,000, at least 20,000, at least 50,000, at least 100,000, at least 500,000 up to 1 million or more) labelled RCA products in the first population and counting at least 1,000 (e.g., at least 5,000, at least 10,000, at least 20,000 or at least 50,000, at least 100,000, at least 500,000 up to 1 million or more) labelled RCA products in a region of the membrane to ensure that differences in copy number can be described with statistical rigour.

In an optional embodiment, PCR primers that hybridize to or are identical to the sites flanking those sequences can be used to amplify cloned fragments (and, optionally, any index sequence in the circular DNA molecule) in the DNA molecule by PCR. In this embodiment, the PCR products can be amplified using the primers. In this embodiment, the quantity of the product can be quantified by any suitable qPCR assay (e.g., Taqman assay) or the like. In another embodiment, the product can be sequenced (with or without amplification). In these embodiments, the number of circular molecules corresponding to each locus can be estimated by counting the number of sequence reads corresponding to the locus (e.g., counting how many sequence reads have specific locus-specific barcode sequences). In some embodiments, if an index sequence is used, the number of circular molecules corresponding to each locus can be counted by determining how many different molecular barcode sequences are associated with each locus-specific barcode sequence.

As will be apparent, in this embodiment, the primers used can be sequences compatible with applications such as Illumina's reversible terminator method, Roche's pyrosequencing method (454), Life Technologies' ligation sequencing method (SOLiD platform), or Life Technologies' Ion Torrent platform. Examples of such methods are described in the following references: Margulies et al. (Nature 2005 437:376–80); Ronaghi et al. (Analytical Biochemistry 1996 242:84–9); Shendure (Science 2005 309:1728); Imelfort et al. (Brief Bioinform. 2009 10:609-18); Fox et al. (Methods Mol Biol. 2009; 553:79-108); Appleby et al. (Methods Mol Biol. 2009; 513:19-39); and Morozova (Genomics. 2008 92:255-64), which are incorporated by reference for a general description of the methods and specific steps of these methods, including all starting products, reagents, and final products for each step.

The test genomic sample can be from a patient suspected of having a disease or condition or facing a risk of having a disease or condition, and the result of step (c) indicates whether the patient or his fetus has the disease or condition. In some embodiments, the disease or condition can be cancer, an infectious disease, an inflammatory disease, transplant rejection, or a chromosomal defect such as trisomy.

As indicated above, in some cases, the sample analyzed using this method can be a sample of cfDNA obtained from blood (e.g., from a pregnant woman's blood). In these embodiments, the method can be used to detect chromosomal abnormalities in a developing fetus (as described above) or to calculate, for example, the fraction of fetal DNA in a sample.

Illustrative copy number abnormalities that can be detected using the method include, but are not limited to, trisomy 21, trisomy 13, trisomy 18, trisomy 16, XXY, XYY, XXX, monosomy X, monosomy 21, monosomy 22, monosomy 16, and monosomy 15. The following table lists other copy number abnormalities that can be detected using the methods of the present invention.

Chromosomal abnormalities and disease associations

X: XO (Turner syndrome)

Y:XXY (Klinefelter syndrome)

Y: XYY (double Y syndrome)

Y: XXX (Trisomy X)

Y: XXXX (quadruple X syndrome)

Y: Xp21 deletion (Duchenne/Becker syndrome, congenital adrenal hypoplasia, chronic myeloid leukemia

budding disease)

Y: Xp22 deletion (steroid sulfatase deficiency)

Y: Xq26 deletion (X-linked lymphoproliferative disorder)

1:1p somatic cell (neuroblastoma)

1: Monosomy (neuroblastoma)

1: Trisomy (neuroblastoma)

2: Monosomy (growth retardation, developmental delay, mental retardation, and minor physical abnormalities)

2: Trisomy 2q (growth retardation, developmental delay, intellectual disability, and minor physical abnormalities)

3: Monosomal (non-Hodgkin's lymphoma)

3: Trisomy 3 (non-Hodgkin lymphoma)

4: Monosomy (acute nonlymphocytic leukemia (ANLL))

4: Trisomy 4 (acute nonlymphocytic leukemia (ANLL))

5:5p (Cri du chat; Lejeune syndrome)

5:5q somatic (myelodysplastic syndrome)

5: Monosomy (myelodysplastic syndrome)

Trisomy 5 (Myelodysplastic Syndrome)

6: Monosomy (clear cell sarcoma)

6: Trisomy 6 (clear cell sarcoma)

7: 7q11.23 deletion (Williams syndrome)

Monosomy 7 (monosomy 7 syndrome in children; somatic: renal cortical adenoma; myelodysplastic syndrome)

Trisomy 7 (monosomy 7 syndrome in children; somatic: renal cortical adenoma; myelodysplastic syndrome)

8: 8q24.1 deletion (Langer-Giedon syndrome)

8: Monosomy (myelodysplastic syndrome; Warkany syndrome; somatic: chronic myeloid leukemia

Blood disease)

8: Trisomy (myelodysplastic syndrome; Warkany syndrome; somatic: chronic myeloid leukemia

Blood disease)

9: Monosomy 9p (Alfi’s syndrome)

9: Monosomy 9p (Rethore syndrome)

9: Partial trisomy (Rethore syndrome)

9: Trisomy (complete trisomy 9; mosaic trisomy 9)

10: Monosomy (ALL or ANLL)

10: Trisomy ALL or ANLL

11:11p- (aniridia; Wilm tumor)

11:11q- (Jacobsen syndrome)

Monosomy 11 (myeloid lineage involvement (ANLL, MDS))

11: Somatic trisomy (myeloid lineage involvement (ANLL, MDS))

12: Monosomy (CLL, juvenile granulosa cell tumor (JGCT))

12: Trisomy 12 (CLL, juvenile granulosa cell tumor (JGCT))

13: 13q- (13q- syndrome; Orbeli syndrome)

13:13q14 deletion (retinoblastoma)

Monosomy 13 (Patau syndrome)

Trisomy 13 (Patau syndrome)

14: Monosomy (myeloid diseases (MDS, ANLL, atypical CML)

14: Trisomy 14: Somatic myeloid diseases (MDS, ANLL, atypical CML)

15:15q11-q13 deletion (Prader-Willi, Angelman syndrome)

15: Monosomy (Prader-Willi, Angelman syndrome)

15: Somatic trisomy (involvement of myeloid and lymphoid lineages, eg, MDS, ANLL, ALL, CLL)

16:16q13.3 deletion (Rubenstein-Taybi)

16: Monosomy (Papillary Renal Cell Carcinoma (Malignant))

16: Trisomy 16 (Papillary Renal Cell Carcinoma (Malignant))

17: 17p-somatic (17P syndrome in myeloid malignancies)

17:17q11.2 deletion (Smith-Magenis)

17: 17q13.3(Miller-Dieker)

17: Monosomy (renal cortical adenoma)

17: Trisomy 17 (renal cortical adenoma)

17: 17p11.2-12 (Charcot-Marie Tooth syndrome type 1; HNPP)

Trisomy 17 (Charcot-Marie Tooth syndrome type 1; HNPP)

18: 18p- (partial monosomy 18p or Grouchy Lamy Thieffry syndrome)

18:18q- (Grouchy Lamy Salmon Landry syndrome)

18: Monosomy (Edwards syndrome)

Trisomy 18 (Edwards syndrome)

19: Monosomy (Edwards syndrome)

Trisomy 19 (Edwards syndrome)

20: 20p- (Trisomy 20p)

20:20p11.2-12 deletion (Alagille)

20:20q- (somatic: MDS, ANLL, polycythemia vera, chronic neutrophilic leukemia)

20: Monosomy (papillary renal cell carcinoma (malignant))

20: Trisomy 20 (Papillary Renal Cell Carcinoma (Malignant))

21: Monosomy (Down syndrome)

21: Trisomy (Down syndrome)

22: 22q11.2 deletion (DiGeorge syndrome, velocardiofacial syndrome, conotruncal-facial anomaly)

Jung's syndrome, autosomal dominant Opitz G/BBB syndrome, Caylor cardiofacial syndrome)

22: Monosomy (complete trisomy 22)

22: Trisomy (complete trisomy 22)

The methods described herein can be used to analyze genomic DNA from virtually any organism (including but not limited to plants, animals (e.g., reptiles, mammals, insects, worms, fish, etc.)), tissue samples, bacteria, fungi (e.g., yeast), bacteriophages, viruses, cadaver tissues, archaeological/ancient samples, etc. In certain embodiments, the genomic DNA used in the method can be derived from a mammal, wherein in certain embodiments, the mammal is a human. In an exemplary embodiment, the genomic sample can contain genomic DNA from a mammalian cell (e.g., a human, mouse, rat, or monkey cell). The sample can be prepared from cultured cells or cells of a clinical sample (e.g., cells of a tissue biopsy, scrapings, or lavage fluid, or cells of a forensic sample (i.e., cells of a sample collected at a crime scene)). In a specific embodiment, a nucleic acid sample can be obtained from a biological sample (e.g., a cell, tissue, body fluid, and feces). Target body fluids include, but are not limited to, serum, plasma, saliva, mucus, phlegm, cerebrospinal fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovial fluid, urine, amniotic fluid, and semen. In specific embodiments, the sample can be obtained from a subject (e.g., a human). In some embodiments, the sample analyzed can be a sample of cfDNA obtained from blood (e.g., from pregnant blood).

For example, in some embodiments, a sample of DNA can be obtained and the sample is digested with one or more restriction enzymes (or RNA-guided endonucleases such as cas9) to produce predictable fragments (the median size of the fragments can be in the range of 20-100 bases). The method described above can be performed on the digested DNA, and using the methods described herein, the number of fragments corresponding to one locus (e.g., a chromosome) can be compared with the number of fragments corresponding to another locus (e.g., another chromosome). As shown, the method can be used to identify copy number differences associated with a disease or condition, for example, chromosomal aneuploidy.

As indicated above, in some cases, the sample analyzed can be a sample of cfDNA obtained from blood (e.g., from a pregnant woman's blood). In these embodiments, the method can be used to detect chromosomal abnormalities in a developing fetus or to, for example, calculate the fraction of fetal DNA in a sample.

Reagent test kit

The present disclosure also provides a kit for implementing the subject method as described above. In certain embodiments, the kit may comprise: (a) a set of splint oligonucleotides of formula X'-A'-B'-Z', wherein: in the set: (i) the sequences of A and B' vary, and (ii) the sequences of X' and Z' are different from each other and are not variable; and in each molecule: (i) sequence A' is complementary to a fragment of a genome and (ii) sequence B' identifies the locus from which the genomic fragment is derived, said genomic fragment hybridizing to the adjacent A' sequence; (b) one or more probes comprising sequences X and Z, wherein: i. sequences X and Z are not variable and hybridize to sequences X' and Z', and (c) a set of locus-specific oligonucleotides of sequence B; and wherein: each splint oligonucleotide of (a) is capable of hybridizing to the probe sequence of (i) (b); (ii) the locus-specific oligonucleotide of (c); and, (iii) the genomic fragment of (a), to produce a ligatable complex of formula X-A-B-Z, wherein sequence B identifies the locus of the adjacent sequence A. In some embodiments, the one or more probes of (b) comprise a first oligonucleotide comprising sequence X and a second oligonucleotide comprising sequence Y. In some embodiments, the kit can also include a PCR primer pair that hybridizes to one or more probes comprising sequences X and Y. In certain embodiments, the one or more probe sequences of (b) are backbone probes of the formula X-Y-Z, and the ligatable complex is a circular ligatable complex of the formula X-A-B-Z-Y, wherein sequence Y connects sequences X and Z, and sequence B identifies the locus of the adjacent sequence A. In these embodiments, the kit can also include a rolling circle amplification primer that hybridizes to a sequence in the backbone probe. In these embodiments, the kit can include a plurality of distinguishably labeled oligonucleotides, wherein each distinguishably labeled oligonucleotide hybridizes to the complement of the B 'sequence. The kit can additionally contain a ligase and/or a strand displacement polymerase for performing rolling circle amplification.

The various components of the kit may be present in separate containers or certain compatible components (eg, the first and second splint probe sets and the first and second locus-specific probes) may be pre-combined into a single container as desired.

In addition to the components mentioned above, the subject kits can also include instructions for using the components of the kit to practice the subject methods.

Example

The following examples are put forth so as to provide those of ordinary skill in the art with additional disclosure and description of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to indicate that the experiments below are all or the only experiments performed.

Example 1

Initial data for validation methods

The purpose of this experiment is to compare the method using a chromosome-specific backbone oligonucleotide (for example, the backbone oligonucleotide used to capture fragments from a first chromosome (for example, chromosome 21) is different from the backbone oligonucleotide used to capture fragments from a second chromosome (for example, chromosome 18), as described in WO 2015083001 and WO 2015083002) with a method in which the same backbone oligonucleotide is used for all chromosomes tested. This is shown in Figure 6. As shown, the "new" design uses chromosome-specific sequences (for example, A or B) cloned into the same circular product as target fragments to determine the source of the cloned fragments. In the new method, a single backbone oligonucleotide is used (compared to the multiple backbone oligonucleotides in the previous method), and the same RCA primer or single PCR primer pair can be used to amplify cloned fragments from all chromosomes.

Cell line DNA (10ng) is digested, denatured and hybridized with " old " design probe and " new " design probe. After hybridization and connection, ligation reaction is accepted exonuclease treatment, to remove any non-circularized DNA in solution. The remaining ring product serves as the template in the RCA reaction, and the RCA reaction produces a concatemer copy of the ring product. These RCA products are labeled with fluorescently labeled oligonucleotides that are complementary to the " splint " sequence, and are deposited on a solid support to detect.

Thirteen cfDNA samples from pregnant women were processed using the same reaction as described above.

For all reactions, the number of each object (RCA product) was counted for each color. For each sample, the ratio of the number of objects in color A/B was calculated and the coefficient of variation was calculated as a measure of the accuracy of the assay. A low coefficient of variation makes it possible to accurately measure samples with low fetal fractions. This was demonstrated by adding samples containing a low spike-in amount of a trisomy 21 cell line sample.

Based on the data shown in Figure 7, the new design produces lower CVs for both cell line DNA and cfDNA, enabling more precise measurement of chromosomally abnormal fetal DNA.

Without wishing to be bound by any particular theory, it is believed that this method may be less sensitive to impurities in the sample.

Example II

Clinical sample analysis

cfDNA samples were prepared from 26 individuals with normal pregnancies and 4 individuals carrying fetuses with trisomy 21. Blood (10 ml) from each patient was centrifuged to separate plasma from red blood cells and a buffy coat. The corresponding plasma (approximately 3-5 ml/patient) was subjected to a bead-based DNA extraction protocol, resulting in extracted cfDNA diluted in 50 μl of buffer.

cfDNA was then processed as described above and analyzed by digitally counting rolling circle products using a fluorescence microscope. All four positive cases were detected with a z-score greater than 3. The CV for normal samples was calculated to be 0.49%, demonstrating the high precision of the assay.

Claims (23)

1. A probe system for analyzing nucleic acid samples, comprising: (a) The set of oligonucleotides labeled with sequence B; (b) A set of splice oligonucleotides of formula X’-A’-B’-Z’, wherein: In this set: (i) sequences A’ and B’ are variable, and (ii) sequences X’ and Z’ are distinct from each other and are not variable; and, In each splice oligonucleotide: (i) sequence A’ is complementary to a genomic fragment of a nucleic acid sample, wherein the genomic fragment is the genomic fragment of sequence A, and (ii) sequence B’ is complementary to at least one member of an oligonucleotide set; Each labeled oligonucleotide or its complementary B' sequence in the splice oligonucleotide identifies (i) the locus in the genome from which the genomic fragment originates or (ii) the chromosome from which the genomic fragment originates; and (c) One or more probe sequences containing X and Z, wherein sequences X and Z are not variable and are capable of hybridizing with sequences X’ and Z’; Each of the splice oligonucleotides can hybridize to: (i) a probe sequence, (ii) a member of the oligonucleotide set and (iii) a genomic fragment, thereby producing a linkable complex of the formula X-A-B-Z. 2. The probe system of claim 1, wherein the labeled oligonucleotide set comprises at least two different B sequence labeled oligonucleotides, and the splice oligonucleotide set contains: at least 100 different A’ sequences; and at least two different B’ sequences complementary to the at least two different labeled oligonucleotides. 3. The probe system of claim 1, wherein each labeled oligonucleotide or its complementary B' sequence in the splice oligonucleotide corresponds to a genomic fragment. 4. The probe system of claim 1, wherein the genomic fragment is derived from a mammalian genome. 5. The probe system of claim 1, wherein each labeled oligonucleotide or its complementary B’ sequence in the splint oligonucleotide identifies one or more of chromosomes 21, 18 and 13. 6. The probe system of claim 1, wherein the genomic fragment is a restriction fragment. 7. The probe system of claim 1, wherein one or more probe sequences of (c) further comprise an oligonucleotide comprising sequence Y, and wherein the linkable complex is linear. 8. The probe system of claim 1 further comprises a PCR primer pair that hybridizes with one or more probes of (c). 9. The probe system according to any one of claims 1-6, wherein one or more probe sequences in (c) comprise a main-chain probe of formula X-Y-Z, wherein Y comprises an oligonucleotide sequence, such that the linkable complex is a cyclic linkable complex of formula X-A-B-Z-Y, wherein sequence Y binds to sequences X and Z. 10. The probe system of claim 9, further comprising rolling circle amplification primers that hybridize with sequences in the main-chain probe. 11. The probe system of claim 9, further comprising: (A) Rolling circle amplification primers that hybridize the sequence with the main-chain probe; and (B) Up to four distinguishably labeled detection oligonucleotides, wherein each distinguishably labeled detection oligonucleotide hybridizes with the B’ sequence. 12. Use of the probe system according to any one of claims 1-11 in the preparation of a kit for a method of determining whether a genomic sample is aneuploid, wherein the method comprises: (a) Hybridizing the probe system according to any one of claims 1-11 with a test genome sample containing a genomic fragment to produce a connectable complex of formula X-A-B-Z; (b) Ligating the ligation complex to produce a product DNA molecule of formula X-A-B-Z; and (c) Count the product DNA molecules corresponding to each sequence B or B’. 13. The use according to claim 12, wherein counting is performed by sequencing the product DNA molecule or its amplification product to produce a sequence readout, and counting the number of sequence readouts containing each sequence B or its complement. 14. The use according to claim 12, wherein the product DNA molecule is circular, and the counting includes amplifying the product DNA molecule by rolling circle amplification and counting the number of amplified products containing each sequence B or its complement. 15. The use according to claim 14, wherein the method comprises labeling the RCA product with a probe that is distinguishably labeled and hybridizes to sequence B’, and counting by counting the number of RCA products for each distinguishable label. 16. The use according to claim 15, further wherein the method comprises: i. depositing RCA products on a planar support; and ii. counting the number of RCA products of each marker in a region of the support. 17. The use according to claim 16, wherein the support is a glass slide. 18. The use according to claim 16, wherein the support is a porous transparent capillary membrane. 19. The use according to any one of claims 12-18, wherein different sequences B and their complementary sequences B’ identify different chromosomes, and the method further comprises comparing the number of product DNA molecules containing the first sequence B or B’ with the number of product DNA molecules containing the second sequence B or B’ to determine whether the genomic sample has aneuploidy. 20. The use according to any one of claims 12-18, wherein the method comprises comparing the counting result of step (c) with the counting result obtained from one or more reference samples. 21. The use according to any one of claims 12-18, wherein the genomic sample being tested is from a patient suspected of having a disease or condition or at risk of having a disease or condition, and the counting result of step (c) provides an indication of whether the patient or their fetus has a disease or condition. 22. The use according to claim 21, wherein the disease or condition is cancer, an infectious disease, an inflammatory disease, transplant rejection, or trisomy. 23. The use according to any one of claims 12-18, wherein the fragment is a limiting fragment.