HK40004594A - Methods for haplotype and diplotype determination - Google Patents

Description

Method for determining haplotype and haplotype

This application has priority to us provisional application 62/601,136 filed on 3/14 of 2017, which is incorporated by reference in its entirety for all purposes herein.

Technical Field

The invention belongs to the field of genetics. In particular, it relates to allelic localization, haplotyping and doubling.

Background

Variations in gene sequence have been recognized at multiple sites on the human genome. Genetic variations or polymorphisms may have functional effects, e.g., some polymorphisms may make an individual more susceptible to disease, or may determine the mode of drug metabolism. Polymorphisms exist in many different forms, including single nucleotide variations, multiple base insertions, microsatellite repeats, dinucleotide repeats, trinucleotide repeats, and sequence rearrangements. Of these sequence polymorphisms, single base variations, also known as Single Nucleotide Polymorphisms (SNPs), are most common in the human genome.

In the human genome, there is about one SNP every three hundred bases. Since humans are diploid organisms, multiple SNPs can be inherited together and appear on one DNA strand, or from parents separately, and thus appear on different copies of the same gene. Phase information of SNPs, i.e., whether they occur on the same strand (cis) or on different strands (trans), is very important because it is well known that phase information affects disease risk, severity of disease phenotype and drug response. Some mutations mask the deleterious effects of another when they are in cis with each other. For example, thrombophilia is associated with two mutation sites in the methylenetetrahydrofolate reductase (MTHFR) gene, with a C (cytosine) to T (thymine) mutation at position 677 (C67 677T) and an a (adenine) to C (cytosine) mutation at position 1298 (a 1298C). However, a pathological phenotype is only observed if the two mutation points are present in trans to each other. In other cases, the effects are compounded when they are present in cis, such as two independent SNPs associated with lung cancer and parkinson's disease. There is increasing evidence that about 1-5% of human genes are expressed in an allele-specific manner, meaning that one copy can be expressed at a different rate than the other, and therefore, if the combination of SNPs affects the function of the corresponding protein, sequence information for each allele is also important.

Although phase information of SNPs is important, this information is often missed in conventional DNA sequencing techniques because DNA fragments are randomly broken before sequencing. PHASE information is typically obtained by processing the results of gene sequencing data from three parents and children through computational and statistical algorithms such as PHASE and hellxtree. However, this method is limited by the accuracy of bioinformatics software and the availability of family data, and phase information of rare mutation sites at low frequency cannot be obtained. Alternatively, a direct laboratory-based approach may be employed. These methods include long fragment sequencing combined with more powerful computational tools, applying traditional sequencing methods to single DNA molecules to detect only cis SNPs, or using contiguous bases to resolve phase information by successive addition of double bases.

The phasing of haplotypes and duplexes typically requires multiple rounds of repeated Polymerase Chain Reactions (PCR) and sequencing of the products of the second (or subsequent) round of PCR to determine which SNPs are co-expressed. The most widely used conventional phasing method is Sanger sequencing, which requires specialized instrumentation and can only measure SNPs over a length of 700 nt.

Current methods for determining haplotypes and duplications are limited by: requiring a large amount of labor, specialized equipment, and high-performance computing equipment to analyze the data.

Therefore, there is an urgent need for a simple, fast method for determining exact allelic information on chromosomal copies, and thus determining haplotypes and diplotypes, which overcomes or at least ameliorates one or more of the disadvantages described above.

Disclosure of Invention

In one aspect, the present application provides a method of determining a haplotype or a haplotype in a genetic sample, the method comprising the steps of: a) contacting a probe complex with the genetic sample, wherein the probe complex comprises at least two probes; b) hybridizing at least two probes to a polynucleotide sequence, wherein each of the plurality of probes is specific for one of two or more genetic variations in the polynucleotide sequence; c) determining the presence or absence of at least one genetic variation by detecting a signal emitted by at least one probe; d) removing or replacing at least one of the probes from the sample; e) detecting the change in the signal to determine the haplotype or the haplotype in the genetic sample.

In one aspect, the present application provides a kit for use in the methods disclosed herein, the kit comprising at least two probes and instructions for use, wherein each of the at least two probes is specific for one of the two or more genetic variants.

Definition of

As used herein, "genetic variation" refers to a variation of one or more nucleotides in a gene sequence relative to a reference nucleotide sequence.

As used herein, the term "haplotype" refers to two or more alleles on a chromosome or a portion of a chromosome. The term haplotype may also refer to two or more Single Nucleotide Polymorphisms (SNPs) on a single chromosome or a portion of a chromosome.

As used herein, the term "doubled" refers to matched haplotype pairs on homologous chromosomes.

As used herein, the term "allele" refers to any of two or more different forms of a gene occupying the same position (locus) on a chromosome.

As used herein, the term "phase" or "phasing" refers to the position of one gene variant or SNP relative to another gene variant or SNP. Two or more genetic variations (SNPs) occurring on the same nucleic acid strand are in cis configuration, while two or more genetic variations (SNPs) occurring on different nucleic acid strands are in trans configuration.

As used herein, the term "hybridize" or other grammatical variant refers to the annealing of a probe to a target nucleotide sequence by non-covalent interactions. It is generally accepted that any hybridization reaction is carried out under stringent conditions. "stringent conditions" refers to any hybridization conditions that allow a probe to specifically bind to a nucleotide sequence but not to any other nucleotide sequence. It is within the ability of the skilled artisan to vary the parameters of the hybridization reaction, such as temperature, probe length and salt concentration, to achieve specific binding hybridization.

As used herein, the term "probe" refers to a molecule designed to bind to a nucleotide sequence and can be used to recognize a particular nucleotide sequence in a target or sample. The probe may include a nucleotide sequence complementary to a particular nucleotide sequence to be identified.

As used herein, the term "foothold" in this context refers to a single-stranded nucleotide sequence that is capable of binding to a given target nucleic acid sequence within a probe. Binding of the foothold sequence to the target sequence triggers separation of the probe strands.

As used herein, the term "polymorphism" refers to the occurrence of two or more alternative genomic sequences or alleles between different genomes or individuals. "polymorphic" refers to the situation where two or more variants of a particular genomic sequence can be found in a population. A "polymorphic site" is a site at which a variation occurs.

As used herein, the term "single nucleotide polymorphism" or "SNP" refers to a single base pair change in a nucleotide sequence. A typical single nucleotide polymorphism is a substitution of one nucleotide for another at a polymorphic site. Single nucleotide deletions or insertions may also lead to single nucleotide polymorphisms. Polymorphic sites may often be occupied by two different nucleotides between different genomes or different individuals.

Drawings

The invention will be better understood by reference to the detailed description and by consideration of non-limiting examples and the accompanying drawings.

FIG. 1 is a schematic representation of a two-step Conditional Displacement Hybridization (CDHA) assay. FIG. 1A shows the binding of two double-stranded probes in the presence of the corresponding SNP sites and the putative fluorescent signals obtained for each target. FIG. 1B shows the second step of CDHA analysis, which involves the addition of a polymerase. The corresponding fluorescence signal is also shown. (TD: target with SNP A and SNP B; TA: target with SNP A, TB: target with SNP B, WT: wild type).

FIG. 2 is a schematic representation of a Conditional Displacement Hybridization Assay (CDHA) based on universal probe design. FIG. 2A shows the expected time course of the change in fluorescence signal for the X-probe and its corresponding target, as well as for the two fluorophores. FIG. 2B shows the TD/WT and TA/TB doublet fluorescence reading based discrimination after addition of polymerase. (TD: target with SNP A and SNP B, TA: target with SNPA, TB: target with SNP B, WT: wild type).

FIG. 3 is a schematic of a magnetic bead mediated conditional separation assay for SNP phasing analysis. FIG. 3A shows the putative fluorescent signals when a three-stranded probe attached to a magnetic bead via a streptavidin-biotin linker reacts with four possible targets. FIG. 3B shows the distinction of the TD/WT and TA/TB diploids when using magnetic beads for separation. (TD: target with SNP A and SNP B, TA: target with SNP A, TB: target with SNP B, WT: wild type).

FIG. 4 is a schematic of phase analysis of three SNPs using Conditional Displacement Hybridization Analysis (CDHA). FIG. 4A is a diagram of different targets with three SNP sites. FIG. 4B shows a general schematic of a three SNP phase assay consisting of two probe wells that will be subjected to the same hybridization steps and conditional permutations by a polymerase. In well 1, probe B has a 3 'modification that prevents polymerase extension reaction, and in well 2, probe A also has the same 3' modification. The footholds and recognition sequences were identical for all three probes in both wells. FIG. 4C shows the fluorescence signal of the target in well 1 and well 2 before addition of polymerase. Further illustrates that there is no change in the fluorescent signal of the haplotype upon addition of the polymerase. This means that samples containing these haplotypes can only be identified in other samples. FIG. 4D shows tabulation of the results, demonstrating that all possible diploid combinations for targets containing three SNP sites can be phased by conditional displacement hybridization analysis.

FIG. 5 is a schematic diagram of three SNP phasing analysis using conditional separation by magnetic bead analysis. FIG. 5A shows two sample wells with different placement of magnetic bead probes, probe C in sample well 1 and probe B in sample well 2. As can be seen in FIG. 5B, after magnetic separation, the haplotype containing SNP C remained in well 1 and the haplotype containing SNP B remained in well 2. The resulting readings of the fluorescence signals from well 1 and well 2 before and after magnetic separation are summarized in fig. 5C and 5D, respectively, and the results show that all possible diploid combinations for targets containing three SNP sites can be phased by the conditional separation assay.

FIG. 6 shows the phasing results for a diploid combination of 10 SNPs. FIG. 6A shows the results of 10 different twofold fluorescence measurements. Each sample containing a specific diploid was incubated with diagnostic probes (for SNP A and SNP B) for 30 minutes followed by addition of polymerase and deoxyribonucleoside triphosphates (dNTPs) for an additional 30 minutes. The fluorescence signal generated in the initial probe hybridization step indicates the number and type of SNPs present on both strands, the dark line corresponding to SNP A and the light line corresponding to SNPB. That is, the duplex type containing two, one or no SNPs indicates fluorescence signals in the ranges of 0.8-1, 0.5-0.7 and 0.1-0.3, respectively. As shown, the fluorescence information can identify eight diplotypes at most, but cannot distinguish the diplotypes formed by TD + WT and TA + TB (as shown in the box), wherein the two diplotypes contain the same number and type of SNPs. Next, it was determined whether these two SNPs were in cis (TD + WT) or trans (TA + TB) configuration by changing the change in the fluorescence signal of SNP A (black line) in the second reaction step. Only the cis (cis) configuration causes a significant decrease in the fluorescence signal (i.e., 0.2-0.3). The colored circles below the fluorescence curves indicate the number of SNPs A and B present on both sets of strands as inferred from the fluorescence values, and the results are summarized in the table of FIG. 6B.

Detailed Description

In one aspect, the present invention relates to a method of determining haplotype or diplotypes in a genetic sample comprising the steps of: a) reacting a probe complex with the genetic sample, wherein the probe complex comprises at least two probes; b) hybridizing at least two probes to the polynucleotide sequence, wherein each of the at least two probes is specific for one of the two or more genetic variations on the polynucleotide sequence; c) determining the presence or absence of at least one genetic variation by detecting a signal emitted by at least one probe, wherein detection of the signal indicates the presence of the genetic variation; d) removing or replacing at least one of the probes from the sample; e) detecting the change in the signal to determine the haplotype or the haplotype in the genetic sample.

In one example, the probe complex can include at least two probes, and the probe complex can also include a double-stranded nucleic acid molecule such as a double-stranded DNA molecule, a triple-stranded nucleic acid molecule such as a triple-stranded DNA molecule, and a quadruplex nucleic acid molecule such as a quadruplex DNA molecule. The design of the probe complexes is determined by the application of the probe complexes, e.g., one probe complex may be designed to be able to diagnose two genetic variations or SNPs in a sample simultaneously. In another example, the probe complex may be designed to contain a universal fluorophore and quencher pair and probes for identifying specific gene variations or SNPs.

In one embodiment, two discrete probes may be hybridized to a connecting strand to form a triple-stranded DNA molecule.

It is generally recognized that each probe can interact with a specific target sequence on a polynucleotide sequence. The polynucleotide sequence may be isolated or purified from genetic samples including, but not limited to, blood, plasma, serum, buccal smears, amniotic fluid, prenatal tissue, sweat, nasal swabs, urine, organs, tissues, debris and cells isolated from mammals including humans. The genetic sample may also include a section of the genetic sample including tissue (e.g., a section of an organ or tissue). In other embodiments, the isolated polynucleotide sequence may be amplified by known methods. In a preferred embodiment, the polynucleotide sequence may be amplified using the Polymerase Chain Reaction (PCR) or an isothermal amplification process.

Each probe may interact with its specific target sequence sequentially or simultaneously. Polynucleotide sequences may include nucleic acids, nucleic acid fragments, plasmids, and other molecules, such as gene fragments, and the like. The probes may be nucleic acids, oligonucleotides, nucleic acid variants, such as Peptide Nucleic Acids (PNAs) or Locked Nucleic Acids (LNAs), peptides, proteins, dyes, fluorophores, magnetic beads, lipids, drugs or small molecules. Any combination of probe types may be used in a given experiment.

In certain embodiments, a probe may comprise one or more nucleotide sequences specific for one or more target sequences.

The probe may also contain a dye, which may refer to a substance used to color or produce a luminescent or fluorescent signal to a material. The dye can absorb or emit light of a particular wavelength and can bind to the probe or target by intercalation, non-covalent binding, or covalent binding. The dye may also be a chemiluminescent or fluorescent molecule. Examples of chemiluminescent molecules include, but are not limited to, N- (4-aminobutyl) -N-ethylisobutol, luminol, coelenterazine, ruthenium complexes, such as Ru (BPS))₃ ^4-(wherein BPS is 4, 7-diphenyl-1, 10-phenanthroline disulfonate or bathophenanthroline disulfonate), Ru (BPS)₂(bipy)^2-(wherein bipy is 2,2' -bipyridine), Ru (BPS) ((bipy))₂And tris (2,2' -bipyridine) ruthenium (II) (Ru (bipy)₃ ²⁺) And ruthenium analogs. The fluorophore may be a protein or peptide, a small organic compound or a synthetic oligomer or polymer, for example, the fluorophore may be a non-protein organic fluorophore selected from the group consisting of: xanthine derivatives, cyanine derivatives, squaraine derivatives and ring-substituted squaraines, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, arylmethyl derivatives and tetrapyrrole derivatives. Other chemiluminescent or fluorescent molecules known in the art may also be suitably employed within the scope of the present invention. It will also be appreciated by those skilled in the art that any set or combination of fluorophores or dyes may be used, but that they should have non-overlapping excitation/emission spectra.

In one embodiment, the probe may further comprise a quencher. A quencher is any molecule or agent that reduces the intensity of chemiluminescence or fluorescence, and one example of a quencher can be an organic or inorganic molecule having a network of conjugated double bonds. Other examples of quenchers include, but are not limited to, molecular oxygen, iodide ions, and acrylamide. In one embodiment, the fluorophore and the quencher may be on different strands of the probe.

In the methods of the invention, the at least two probes may comprise at least two different fluorophores. The at least two probes may also contain at least two quenchers, which may be the same or different.

In further embodiments, one or more quenchers may be added to the connecting strands hybridized to the at least two discrete probes. The linker can be covalently modified at the 5 'or 3' end of the linker with one or more quencher molecules.

In another embodiment, one of the at least two probes may further comprise a magnetic bead. The magnetic beads are linked to one or more of the at least two probes via streptavidin molecules. In one embodiment, the magnetic beads are streptavidin-modified magnetic beads that are bound to the probes by biotin modification in the probes. In another embodiment, the magnetic beads may be covalently bound to the probes by activating functional groups on the surface of the magnetic beads, such as N-hydroxysuccinimide (NHS), and reacting the magnetic beads with probes comprising one or more amine-functionalized oligonucleotides.

In certain embodiments, the probes may be immobilized on a surface, which may be a solid surface or a substrate. Examples of surfaces include, but are not limited to, gold or silicon, films such as eggshell membrane (ESM), polymeric substrates, or gels. In certain embodiments, the probe may be attached to the solid surface by a gold-thiol-DNA bond, a silicon-NHS-amine-DNA interaction, or a polymer-streptomycin avidin-biotin-DNA interaction.

Hybridization of a probe to a nucleotide sequence can be achieved by any means of annealing the probe to the nucleotide sequence. In one embodiment, hybridization can be achieved by foothold-mediated strand displacement. Wherein the hybridization reaction is triggered by a foothold sequence on the probe that anneals to a complementary sequence on the polynucleotide sequence. Annealing of the foothold sequence on the probe to the polynucleotide sequence can result in separation of the strands of the probe and hybridization of the probe strand comprising the foothold sequence to the polynucleotide sequence. Strands that do not contain the pivot sequences are replaced. It is generally understood that the length of the probe sequence and/or the region containing the footholds thermodynamically determines the specificity of hybridization of the probe to the polynucleotide strand.

Hybridization of the probe to the polynucleotide sequence can cause the probe to signal. Detecting the presence of the emitted signal may indicate the presence of a genetic variation. In other embodiments, the intensity of the emitted signal is measured to determine the presence or copy number of a genetic variation in the genetic sample. The strength of the transmitted signal may be measured based on the reference signal. The reference signal may be a signal emitted from a genetic sample with a known genetic variation, e.g., a wild-type sample, or the reference signal may be a signal emitted from the same genetic sample prior to addition of an enzyme or prior to removal or replacement of one or more probes. The reference signal can also be a signal emitted from a genetic sample without hybridization of the probe to the polynucleotide sequence.

As described herein, the presence or absence of a genetic variation can be determined by detecting a signal emitted by at least one probe using the methods of the invention. The methods of the invention also allow for determining the phasing of at least two or more genetic variations by removing or replacing at least one of the probes from a sample.

In one embodiment, at least one probe may be removed by magnetic separation. Magnetic separation can be used to separate probes attached to magnetic beads from probes not attached to magnetic beads.

In another embodiment, at least one probe may be removed from the sample by a washing step. In one embodiment, at least one probe may be immobilized on a surface, and after hybridization of the target nucleic acid to the immobilized probe, any probe that is not immobilized on the surface after the hybridization step may be removed from the sample by washing.

In another embodiment, at least one probe may be displaced from the sample by the action of a polymerase. In certain embodiments, a probe that binds to a polynucleotide sequence can serve as a primer for a polymerase, and extension of the primer by the polymerase can displace another probe that binds to a polynucleotide sequence.

In a preferred embodiment, the polymerase may be a high fidelity DNA polymerase. The high fidelity DNA polymerase may have no 5 'to 3' exonuclease activity.

In one embodiment, one of the at least two probes contains a modification that prevents extension by a polymerase. One example of a modification that prevents polymerase extension is an overhang, e.g., the overhang may comprise a 3' poly a tail. The overhang region can also comprise a hairpin structure. In a preferred embodiment, one of the at least two probes may be modified with a 3' poly A tail.

Removal or replacement of at least one probe from the genetic sample may result in a change in the presence or intensity level of one or more signals emitted from the genetic sample. Detection of changes in the emitted signal or signals can be used to determine haplotypes or duplications in the genetic sample. In one example, detection of a decrease in the emission signal can indicate that the two genetic variations are present in the cis configuration. In another example, detection of a decrease in the emitted signal may indicate that the two gene mutations are present in the trans configuration.

The methods of the invention can be used to determine the haplotype or diplotype of two or more genetic variations. The two or more genetic variations may be less than 1kb, or greater than 1kb, from each other. It is generally recognized that two or more genetic variations may exist at distances up to the length of a chromosome. For example, two or more genetic variants can be at least 100 nucleotides (nt), 200nt,300nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, 1500nt, or 2000nt from each other. In one embodiment, the two or more genetic variations are located on a chromosome. In a preferred embodiment, two or more genetic variations are at least 700nt apart.

The invention also provides a probe complex for use in the method of the invention comprising at least two probes, a fluorophore and a quencher.

In one embodiment, the probe complex may further comprise a linker that hybridizes to at least two probes. In another embodiment, the probe complexes further comprise magnetic beads attached to one or more of the at least two probes.

The invention also provides a kit for use in the methods of the invention, the kit comprising at least two probes and instructions for use, wherein each of the at least two probes is specific for one of two or more genetic variations.

In one embodiment, the kit may further comprise a polymerase.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be read expansively and without limitation. Also, the terms and expressions which have been employed herein are used as terms of description and not of limitation. There is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it is believed that while the present invention has been particularly disclosed, by way of preferred embodiments and optional features, modification and variation of the present invention may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

The invention has been described broadly and broadly herein and each of the narrower and narrower groups that fall within the broader disclosure also form a part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Experimental part

Non-limiting examples of the present invention and comparative examples will be described in more detail with reference to specific examples and should not be construed as limiting the scope of the invention in any way.

Example 1

Conditional displacement by polymerase

A two-step reaction is involved, where the presence of two SNPs is first diagnosed and if both are present, phase information is deduced by a second set of nucleic acid reactions (see fig. 1). Synthetic target DNA was designed containing one, two or no SNP sites of the two possible SNP sites labeled A and B, separated by 100 nt. Subsequently, two double-stranded DNA (dsdna) probes comprising fluorophore-quencher pairs are added, each probe diagnosing one SNP site in the target DNA. If the SNP is present, the DNA fragment hybridizes to the fluorophore-containing strand of the first probe and displaces the second quencher-containing strand by a foothold mediated strand displacement reaction. The specificity of the reaction is thermodynamically controlled by the probe sequence and the length of the foothold region. Similarly, a second probe containing a different fluorophore is used to diagnose the presence of a second SNP. In solution, the generation of two fluorescent signals cannot distinguish between cis and trans configuration SNPs. Thus, a high fidelity DNA polymerase without 5 'to 3' end exonuclease activity was added. The bound first probe is extended by a polymerase as a primer, thereby displacing a second probe downstream of the first probe. This decrease in fluorescence signal is indicative of cis SNP configuration, which cannot occur when SNPs are located on different strands.

Example 2

Polymerase conditional displacement reactions using universal probes

The above embodiment is modified by using a general-purpose probe (which is called an "X-probe" because of its shape as shown in fig. 2). Firstly, four DNA strands are hybridized to prepare a probe, wherein the four DNA strands comprise a fluorescence labeling strand, a quencher labeling strand and two sequence specificity strands. To phase pairs of SNPs, the same fluorescently labeled strand and quencher-labeled strand are used, but the corresponding sequence-specific strands need to be adjusted. Phase assays were performed on pairs of SNPs using 96-well plates by loading different X-probes in each well and then using the X-probes obtained to perform the same protocol as in the previous section in one assay (FIG. 2). Since the fluorophore-quencher pairs are similar, the same two fluorescence channels are used. This method achieves higher reaction throughput and is a more efficient method for phasing two or more pairs of SNPs simultaneously.

Example 3

Conditional separation by magnetic separation

The invention also provides a method that does not require enzymes. Instead, two fluorescently labeled probes used to diagnose two different SNP sites (referred to as SNP A and SNPB) are first bound to a linker chain, both the 5 'and 3' ends of which are covalently linked to a quencher molecule. The triplex probe is attached to streptavidin-modified magnetic beads via biotin modification in a fluorescently labeled probe. The fluorescent signal is measured twice-the first time after incubation of the bead-attached probes with the appropriate target DNA, and the second time after separation, washing and reconstitution (to the same volume) of the beads. In the first measurement, no signal is shown without target sequence (or with wild-type target sequence present), whereas fluorophores can be used for signal detection when the target sequence contains any SNP. When both fluorophores are present, both fluorophores will emit a signal (FIG. 3). At this step, the fluorescence signal spectra in the presence of diploid TD/WT and TA/TB produced a faint fluorescence signal. Considering that only one fluorescent label strand is attached to one magnetic bead, only one fluorescent signal remains when each SNP is on a different single strand during magnetic separation, while both fluorescent signals remain when both SNPs are on the same single strand (FIG. 3).

Example 4

Phasing of three SNPs by polymerase conditional displacement

The method described above in example 1 was extrapolated to phase three SNP sites. Phasing multiple SNP sites can increase the variety of diseases and conditions that can be identified and expand the possibilities for application of the technology. In this case, two reaction vessels containing the same sample (containing any one, two, or three SNPs) were incubated with three fluorophore-quencher pairs (probes A, B and C) to diagnose the presence of three SNPs, SNP a, SNP B, and SNP C, respectively. The wavelength of the generated fluorescent signal indicates the presence of the corresponding SNP (FIG. 4). In order to derive phase information, slightly modified probes A and B were used in each reaction vessel. In the first sample well, probe B is used with a 3' poly A tail attached so that only probe A can be extended when polymerase is added. Similarly, in a second sample well, probe A was used with a 3' poly A tail attached (panel B). Thus, upon addition of polymerase, a decrease in fluorescence signal in the first sample well provides phase information for SNP B and C relative to SNP a, while a decrease in fluorescence signal in the second sample well provides phase information for SNP C relative to SNP B (fig. 4C). Two different reactions are required since some diploids can only be identified in one of the two sample wells, i.e. more than one diploid in one sample well can produce the same fluorescence signal after the reaction has taken place. By performing two different reactions in sample well 1 and sample well 2, the method ensures that even if the two (or more) diploids have the same fluorescence characteristics in sample well 1, there will be different fluorescence characteristics in sample well 2. Thus, two sample wells are required to ensure that all diploids can be distinguished. The phase information of the three SNPs can be analyzed using the combined information of the two sample wells (fig. 4D).

EXAMPLE 5

Phasing three SNPs Using conditional separation of magnetic particles

Similar to example 4, the conditional separation method using magnetic particles was extrapolated to be used for diagnosis and phasing of three SNPs. This requires two reaction vessels, where magnetic beads are attached to the probes C of the first sample well and to the probes B of the second sample well (FIG. 5).

In the first sample well, the magnetically separated fluorescence spectrum provides phase information relative to probe C, while the second sample well provides phase information relative to probe B (fig. 5B). As shown in example 4, performing two different reactions (in sample well 1 and sample well 2) ensures that even if two (or more) diploids have the same fluorescence characteristics in sample well 1, they will have different fluorescence characteristics in sample well 2. Using the combined information of the two sample wells, the phase information of the three SNPs can be resolved (fig. 5C and 5D).

Example 6

Ten twofold combined phase information

Conditional displacement analysis was performed on 10 possible diploids using the four templates (TD, TA, TB and WT) shown in fig. 6. These 10 diploids are symbolized as fully recorded, semi-recorded and unrecorded (i.e. two, one or no colored circles) according to their fluorescence values at two stages of saturation, and all 10 diploids present their own unique barcode according to their SNP number and phase information. The green and red fluorescence values were quantitatively related to the amount of SNPs A and B prior to addition of polymerase. For example, when there are 2 TD strands, 2 TB strands, or a combination of TD and TB, the green fluorescence value is-1, and these combinations all contain two SNP B, which are symbolized as two green circles. The combinations of TD and TA, TD and WT, TB and TA or TB and WT all contained one SNP B with a fluorescence value of-0.6, represented by a green circle. Finally, the fluorescence signal for those strands without SNP B (e.g., 2 TA strands, 2 WT strands, or a combination of TA and WT) is-0.2, represented by two open green circles. These notations apply equally to the red fluorescence channel. Using this labeling method, it has not been possible to distinguish TD and WT mixtures from TA and TB mixtures, since they produce a half red-green fluorescence signal. But can be determined by a second partial analysis with the addition of a polymerase. The red fluorescence signal remained almost unchanged when TA and TB were present, but was reduced by one third for TD and WT. The fluorescence decrease for other SNP combinations was also consistent.

The above examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will be obvious that numerous modifications and variations of the specific embodiments of the present invention described above and illustrated in the examples may be made without departing from the underlying principles of the invention. All such modifications and variations are intended to be covered by this application.

Claims (24)

1. A method of determining a haplotype or a haplotype in a genetic sample comprising the steps of:

a) contacting a probe complex with a genetic sample, wherein the probe complex comprises at least two probes;

b) hybridizing the at least two probes to a polynucleotide sequence, wherein each of the at least two probes is specific for one of two or more genetic variations in the polynucleotide sequence;

c) determining the presence or absence of at least one genetic variation by detecting a signal emitted by at least one probe, wherein detection of the signal indicates the presence of the genetic variation;

d) removing or replacing at least one of the probes from the sample; and

e) detecting the signal change to determine the haplotype or the haplotype in the genetic sample.

2. The method of claim 1, wherein the probe complex comprises a double-stranded DNA (dsdna) molecule, a triple-stranded DNA molecule, or a quadruple-stranded DNA molecule.

3. The method of claim 2, wherein the probe complex comprises a triple-stranded DNA molecule comprising a first probe and a second probe hybridized to a connecting strand, wherein the first probe and the second probe are separated from each other.

4. The method of any one of claims 1-3, wherein each of the at least two probes comprises a different fluorophore.

5. The method of claim 4, wherein each of the at least two probes further comprises a quencher.

6. The method of claim 5, wherein the fluorophore and the quencher are on different strands of the probe.

7. The method of claim 6, wherein the quencher is on the linker.

8. The method of any one of claims 1-7, wherein one of the at least two probes further comprises a magnetic bead.

9. The method of claim 8, wherein the magnetic bead is a streptavidin-modified magnetic bead that is functionally attached to the probe via a biotin modification in the one probe.

10. The method of any one of claims 1-9, wherein the probe is immobilized on a solid surface.

11. The method of claim 10, wherein the solid surface is a gold substrate, a silicon substrate, or a polymer substrate.

12. The method of any one of claims 1-11, wherein at least one probe is hybridized by foothold-mediated strand displacement.

13. The method of claims 1-12, wherein the at least one probe is removed from the sample by magnetic separation.

14. The method of claims 1-12, wherein the at least one probe is displaced by the action of a polymerase.

15. The method of claim 14, wherein the polymerase is a high fidelity DNA polymerase without 5 'to 3' exonuclease activity.

16. The method of claim 1, wherein one of the at least two probes further comprises a modification that prevents polymerase extension.

17. The method of any one of claims 1-16, wherein the detection of the signal or change in the signal further comprises detecting an intensity of the signal or a change in the intensity of the signal relative to a reference signal to determine the haplotype or diplotype in the genetic sample.

18. The method of claim 17, wherein the reference signal is a signal detected from a known haplotype or diploid.

19. The method of any one of claims 1-18, wherein the presence or absence of two or more genetic variations is determined simultaneously.

20. A probe complex for use in the method of any one of claims 1-19, said probe complex comprising at least two probes, a fluorophore and a quencher.

21. The probe complex of claim 20, further comprising a linker hybridized to the at least two probes.

22. The probe complex of claim 21, further comprising a magnetic bead attached to one or more of the at least two probes.

23. A kit for use in the method of any one of claims 1-19, the kit comprising at least two probes and instructions for use, wherein each of the at least two probes is specific for one of two or more genetic variations.

24. The kit of claim 23, further comprising a polymerase.