Detection of Neighboring Variants

Abstract

The present invention relates to methods, kits, probes, and systems for distinguishing between nucleotide variants that are close in proximity on a gene. The methods, kits, probes, and systems can include the use of a small amplicon assay in combination with two unlabeled probes in a high resolution thermal melting analysis of a biological sample containing a locus of interest in order to discern between disease-causing and benign variants that are close in proximity on a gene within the biological sample. The present invention also relates to method of detecting a disease in a patient based on the patient's genotype by determining whether the patient has a disease-causing variant at a locus of interest. The signature melt curves produced by the unlabeled probe tests can be analyzed using HRMA software to distinguish between disease-causing and benign variants that are close in proximity on a gene within the biological sample.

1 . A method of distinguishing between at least two nearby neighbor variants on a nucleic acid having a locus of interest comprising:
 (a) providing a first aliquot of said nucleic acid having a locus of interest;   (b) incubating said first aliquot of said nucleic acid with a limiting primer, an excess primer, and a first probe that is designed to hybridize to said locus of interest on a target strand of said nucleic acid;   (c) performing asymmetric PCR using said first aliquot to produce an excess of amplicons corresponding to the target strand to which the first probe hybridizes, thereby producing a first probe element;   (d) providing a second aliquot of said nucleic acid target having a locus of interest;   (e) incubating said second aliquot of said nucleic acid target with said limiting primer, said excess primer, and a second probe that is designed to hybridize to said locus of interest on the target strand, wherein said first probe differs in sequence from said second probe;   (f) performing asymmetric PCR using said second aliquot to produce an excess of amplicons corresponding to the target strand to which the second probe hybridizes, thereby producing a second probe element;   (g) generating a first melting curve for the first probe element in a first mixture with a saturating binding dye by measuring fluorescence from said dye as the first mixture is heated;   (h) generating a second melting curve for the second probe element in a second mixture with said saturating binding dye by measuring fluorescence from said dye as the second mixture is heated; and   (i) analyzing said first melting curve and said second melting curve to distinguish between said at least two nearby neighbor variants, wherein a melting signature curve of each of said at least two nearby neighbor variants is different in said first and second melting curves.   
     
     
         2 . The method of  claim 1 , wherein said one or both of said first and second probes are unlabeled. 
     
     
         3 . The method of  claim 1 , wherein steps (a)-(f) are performed simultaneously. 
     
     
         4 . The method of  claim 1 , wherein steps (a)-(f) are performed sequentially. 
     
     
         5 . The method of  claim 1 , wherein said first probe and said second probe each have a sequence that is complementary to a wild-type sequence of the gene. 
     
     
         6 . The method of  claim 1 , wherein the limiting primer and the excess primer are each set close to the variants to reduce the amplicon size for high genotyping sensitivity. 
     
     
         7 . The method of  claim 1 , wherein each of said first and second probes has one or more base pair mismatches at the locus of interest. 
     
     
         8 . The method of  claim 7 , wherein each of said first and second probes has 2 to 5 base pair mismatches at the locus of interest. 
     
     
         9 . The method of  claim 1 , wherein each of said first and second probes, independently, has 2 to 5 base pairs at its 5′-end prior to the locus of interest. 
     
     
         10 . The method of  claim 1 , wherein the first probe has 2 or 3 base pairs at its 5′-end prior to the locus of interest. 
     
     
         11 . The method of  claim 1 , wherein the second probe has 5 base pairs at its 5′-end prior to the mutation. 
     
     
         12 . The method of  claim 1 , wherein the Tm of each of the first and second probes is less than about 5 degrees lower than the Tms of the limiting and excess primers and the difference of the limiting primer's Tm and the excess primer's Tm is less than about 1° C. 
     
     
         13 . The method of  claim 1 , wherein said first probe and said second probe are 34 to 37 bp in length. 
     
     
         14 . The method of  claim 1 , wherein said first probe and said second probe are blocked at their 3′ ends. 
     
     
         15 . The method of  claim 1 , wherein said locus of interest is on a gene associated with a disorder selected from the group consisting of Cystic Fibrosis, Factor V Leiden, human platelet antigens, a RET proto-oncogene associated disease, lactase hemorrhagic telangiectasia, and hereditary hemorrhagic telangiectasia. 
     
     
         16 . The method of  claim 1 , wherein said locus of interest is Exon 11 of Cystic Fibrosis transmembrane conductance regulator gene. 
     
     
         17 . The method of  claim 1 , wherein said locus of interest is Exon 10 of Cystic Fibrosis transmembrane conductance regulator gene. 
     
     
         18 . The method of  claim 17 , wherein said limiting primer has a nucleotide sequence of 5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1). 
     
     
         19 . The method of  claim 17 , wherein said excess primer has a nucleotide sequence of 5′-GT TGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2). 
     
     
         20 . The method of  claim 17 , wherein said first probe has a nucleotide sequence of 5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO:3). 
     
     
         21 . The method of  claim 20 , wherein said first unlabeled probe is blocked at its 3′ end. 
     
     
         22 . The method of  claim 17 , wherein said second probe has a nucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 4). 
     
     
         23 . The method of  claim 22 , wherein said second unlabeled probe is blocked at its 3′ end. 
     
     
         24 . The method of  claim 17 , wherein said at least two nearby neighbor variants are ΔI507 and F508C. 
     
     
         25 . A method of distinguishing between at least two nearby neighbor variants on a gene comprising:
 (a) mixing a first portion of a target nucleic acid having a locus of interest with a first primer and a second primer, the primers configured for amplifying the target nucleic acid having a locus of interest, and a first unlabeled probe;   (b) in parallel, mixing a second portion of said target nucleic acid having a locus of interest with said first primer, said second primer, and a second unlabeled probe;   (c) simultaneously amplifying the target nucleic acid having a locus of interest to generate amplicons that hybridize to said first unlabeled probe and to said second unlabeled probe to form a first probe element and a second probe element, respectively, wherein said first unlabeled probe differs in sequence from said second unlabeled probe;   (d) generating a first melting curve for the first probe element in the presence of a saturating binding dye by measuring fluorescence from said dye as the mixture is heated;   (e) generating a second melting curve for the second probe element in the presence of said saturating binding dye by measuring fluorescence from said dye as the mixture is heated; and   (f) analyzing said first melting curve and said second melting curve to distinguish between said at least two nearby neighbor variants, wherein a probe melting signature curve of each of said at least two nearby neighbor variants is different in said first and second melting curves.   
     
     
         26 . A method of detecting a disease in a patient based on said patient's genotype and a priori knowledge of benign and disease-causing variant gene sequences associated with said disease, comprising:
 (a) obtaining a biological sample from said patient;   (b) subjecting a first portion of said biological sample to asymmetric PCR involving a limiting primer, an excess primer, and a first probe to produce a first probe-amplicon element;   (c) subjecting a second portion of said biological sample to asymmetric PCR involving said limiting primer, said excess primer, and a second probe to produce a second probe-amplicon element;   (d) generating a first melting curve and a second melting curve by subjecting said first and second probe-amplicon melting elements to high resolution thermal melting analysis, respectively;   (e) distinguishing between a benign variant and a disease-causing neighbor variant by analyzing said first melting curve and said second melting curve, wherein a probe melting signature curve of said benign variant and a probe melting signature curve of said disease-causing variant in said first and second melting curves are different; and   (f) determining whether said patient has a disease-causing variant.   
     
     
         27 . The method of  claim 26 , wherein said disease is selected from the group consisting of Cystic Fibrosis, Factor V Leiden, human platelet antigens, a RET proto-oncogene associated disease, lactase hemorrhagic telangiectasia, and hereditary hemorrhagic telangiectasia. 
     
     
         28 . The method of  claim 27 , wherein said disease cystic fibrosis. 
     
     
         29 . The method of  claim 26 , wherein said benign and said disease-causing variant are neighboring variants. 
     
     
         30 . The method of  claim 29 , wherein said benign variant is F508C and said disease-causing variant is ΔI507. 
     
     
         31 . The method of  claim 26 , wherein said limiting primer has a nucleotide sequence of 5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1). 
     
     
         32 . The method of  claim 26 , wherein said excess primer has a nucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2). 
     
     
         33 . The method of  claim 26 , wherein said first probe has a nucleotide sequence of 5′-AAAATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 3). 
     
     
         34 . The method of  claim 33 , wherein said first probe is blocked at its 3′ end. 
     
     
         35 . The method of  claim 26 , wherein said second probe has a nucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATGAATATAG-3′ (SEQ ID NO: 4). 
     
     
         36 . The method of  claim 35 , wherein said second probe is blocked at its 3′ end. 
     
     
         37 . A primer having a nucleotide sequence of 5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1). 
     
     
         38 . A primer having a nucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2). 
     
     
         39 . A probe having a nucleotide sequence of 5′-AAAATATCATCTTTGGTGTTTCCTATG ATGAATATAG-3′ (SEQ ID NO: 3) and being blocked at its 3′ end. 
     
     
         40 . A probe having a nucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCTATGATG AATATAG-3′ (SEQ ID NO: 4) and being blocked at its 3′ end. 
     
     
         41 . A kit comprising:
 (a) a primer having a nucleotide sequence of 5′-GGATTATGCCTGGCACCATTA-3′ (SEQ ID NO: 1);   (b) a primer having a nucleotide sequence of 5′-GTTGGCATGCTTTGATGACG-3′ (SEQ ID NO: 2);   (c) a probe having a nucleotide sequence of 5′-AAAATATCATCTTTGGTGTTT CCTATGATGAATATAG-3′ (SEQ ID NO: 3) and being blocked at its 3′ end;   (d) a probe having a nucleotide sequence of 5′-ATATCATCTTTGGTGTTTCCT ATGATGAATATAG-3′ (SEQ ID NO: 4) and being blocked at its 3′ end; and   (e) instructions for performing a diagnostic test for detecting cystic fibrosis transmembrane conductance regulator Exon 10 variants using a biological sample from a patient.   
     
     
         42 . A method of detecting a disease in a patient based on said patient's genotype and a priori knowledge of benign and disease-causing variant gene sequences associated with said disease, comprising:
 (a) obtaining a biological sample from said patient;   (b) dividing said biological sample into a first portion and a second portion;   (c) performing asymmetric PCR in order to produce a small amplicon in each of said first portion and said second portion;   (c) subjecting said first portion to a first unlabeled probe assay to produce a first melting curve;   (d) subjecting said second portion to a second unlabeled probe assay to produce a second melting curve;   (e) distinguishing between a benign variant and a disease-causing neighbor variant by comparing said first melting curve and said second melting curve, wherein a probe melting signature curve of said benign variant and a probe melting signature curve of said disease-causing variant in said first and second melting curves are different; and   (f) determining whether said patient has a disease-causing variant.   
     
     
         43 . The method of  claim 42 , wherein said first unlabeled probe assay comprises hybridizing a first unlabeled probe to a locus of interest on said small amplicon to form a first probe element, adding a saturated dye to said first probe element to form a mixture, and generating a first melting curve for the first probe element by measuring fluorescence from said dye as the mixture is heated. 
     
     
         44 . The method of  claim 42 , wherein said second unlabeled probe assay comprises hybridizing a second unlabeled probe to a locus of interest on said small amplicon to form a second probe element, adding a saturated dye to said second probe element to form a mixture, and generating a second melting curve for the second probe element by measuring fluorescence from said dye as the mixture is heated. 
     
     
         45 . The method of  claim 42 , wherein said disease is selected from the group consisting of Cystic Fibrosis, Factor V Leiden, human platelet antigens, a RET proto-oncogene associated disease, lactase hemorrhagic telangiectasia, and hereditary hemorrhagic telangiectasia. 
     
     
         46 . A method of designing primers and probes that are useful for thermal melt analysis of a nucleic acid having a locus of interest that contains one or more benign variants and one or more disease-causing variants that are in close proximity, the method comprising:
 (a) selecting a locus of interest on a nucleic acid that is associated with a disease, in which the nucleic acid has one or more benign variants and one or more disease-causing variants in close proximity on said locus of interest;   (b) designing a pair of primers for use in asymmetric PCR, wherein the primers have a Tm difference of less than about 1° C. and are selected to produce an amplicon of about 60-120 base pairs upon amplification of the nucleic acid having a locus of interest; and   (c) designing at least two probes for hybridizing to one strand of the nucleic acid having a locus of interest, wherein the nucleotide sequence of each probe is complementary to the nucleic acid's wild-type sequence, wherein each probe has a Tm that is less 5° C. lower than the Tms of the primers, wherein each probe overlaps the nucleotide positions of the one or more benign and one or more disease-causing variants that are in close proximity on said locus of interest and wherein the probes differ in length.   
     
     
         47 . The method of  claim 46 , wherein the probes differ in length on the 5′ end of the probe. 
     
     
         48 . The method of  claim 46 , wherein the probes have a 3′ end block to prevent extension. 
     
     
         49 . The method of  claim 46 , wherein the length of each of said probes is about ⅓ the length of said amplicon. 
     
     
         50 . A method of distinguishing between at least two nearby neighbor variants on a gene comprising:
 (a) providing an amplicon having a locus of interest;   (b) hybridizing a first unlabeled probe to said locus of interest on a first portion of the amplicon to form a first probe element;   (c) hybridizing a second unlabeled probe to said locus of interest on a second portion of the amplicon to form a second probe element, wherein said first unlabeled probe differs in sequence from said second unlabeled probe;   (d) generating a first melting curve for the first probe element in a first mixture with a saturating binding dye by measuring fluorescence from said dye as the first mixture is heated;   (e) generating a second melting curve for the second probe element in a second mixture said saturating binding dye by measuring fluorescence from said dye as the second mixture is heated; and   (f) analyzing said first melting curve and said second melting curve to distinguish between said at least two nearby neighbor variants, wherein a melting signature curve of each of said at least two nearby neighbor variants is different in said first and second melting curves.   
     
     
         51 . The method of  claim 50 , wherein said amplicon is produced by mixing a target nucleic acid having a locus of interest with a first primer and a second primer, the primers configured for amplifying the target nucleic acid having a locus of interest, and amplifying the target nucleic acid having a locus of interest to generate an amplicon. 
     
     
         52 . The method of  claim 50 , wherein said amplicon is produced using asymmetric PCR. 
     
     
         53 . The method of  claim 50 , wherein steps (a)-(f) are performed simultaneously. 
     
     
         54 . The method of  claim 50 , wherein steps (a)-(f) are performed sequentially. 
     
     
         55 . The method of  claim 50 , wherein said first unlabeled probe and said second unlabeled probe each have a sequence that is complementary to a wild-type sequence of the gene. 
     
     
         56 . The method of  claim 50 , wherein the first primer and the second primer are each set close to the variants to reduce the amplicon size for high genotyping sensitivity. 
     
     
         57 . The method of  claim 50 , wherein the probe has one or more base pair mismatches at the locus of interest. 
     
     
         58 . The method of  claim 50 , wherein the unlabeled probe has 2 to 5 base pairs at its 5′-end prior to the locus of interest. 
     
     
         59 . The method of  claim 50 , wherein the first unlabeled probe has 2 or 3 base pairs at its 5′-end prior to the locus of interest. 
     
     
         60 . The method of  claim 50 , wherein the second unlabeled probe has 5 base pairs at its 5′-end prior to the mutation. 
     
     
         61 . The method of  claim 50 , wherein the probe Tm is less than about 5 degrees lower than primer Tms and the difference of the first primer's Tm and the second primer's Tm is less than about 1° C. 
     
     
         62 . The method of  claim 50 , wherein said first probe and said second probe are 34 to 37 bp in length. 
     
     
         63 . The method of  claim 50 , wherein said first probe and said second probe are blocked at their 3′ ends. 
     
     
         64 . The method of  claim 50 , wherein the first primer is a limiting primer in the asymmetric PCR. 
     
     
         65 . The method of  claim 50 , wherein the second primer is an excess primer in the asymmetric PCR. 
     
     
         66 . The method of  claim 50 , wherein said locus of interest is Exon 10 of Cystic Fibrosis transmembrane conductance regulator gene. 
     
     
         67 . The method of  claim 50 , wherein said locus of interest is Exon 11 of Cystic Fibrosis transmembrane conductance regulator gene. 
     
     
         68 . A method of detecting a disease in a patient based on said patient's genotype and a priori knowledge of benign and disease-causing variant gene sequences associated with said disease, comprising:
 (a) obtaining a biological sample from said patient;   (b) subjecting said sample to asymmetric PCR to produce a small amplicon;   (c) subjecting a first portion of said small amplicon to a first unlabeled probe assay to produce a first melting curve;   (d) subjecting a second portion of said small amplicon to a second unlabeled probe assay to produce a second melting curve;   (e) distinguishing between a benign variant and a disease-causing neighbor variant by analyzing said first melting curve and said second melting curve, wherein a probe melting signature curve of said benign variant and a probe melting signature curve of said disease-causing variant in said first and second melting curves are different; and   (f) determining whether said patient has a disease-causing variant.   
     
     
         69 . A method of distinguishing between at least two nearby neighbor variants on a nucleic acid having a locus of interest comprising:
 (a) performing asymmetric PCR using a primer pair and a first unlabeled probe;   (b) performing asymmetric PCR using said primer pair and a second unlabeled probe, wherein said first unlabeled probe differs in sequence from said second unlabeled probe;   (c) generating a first melting curve for products produced in the asymmetric PCR using the first unlabeled probe in a first mixture with a saturating binding dye by measuring fluorescence from said dye as the first mixture is heated;   (d) generating a second melting curve for products produced in the asymmetric PCR using the second unlabeled probe in a second mixture with said saturating binding dye by measuring fluorescence from said dye as the second mixture is heated; and   (e) analyzing said first melting curve and said second melting curve to distinguish between said at least two nearby neighbor variants, wherein a melting signature curve of each of said at least two nearby neighbor variants is different in said first and second melting curves.   
     
     
         70 . The method of  claim 69 , wherein each of said asymmetric PCRs in steps (a) and (b) produce PCR products comprising small double-stranded amplicons and probe/primer amplicons. 
     
     
         71 . The method of  claim 70 , wherein if two neighboring mutations cannot be clearly separated from melt signatures produced by said probe/primer amplicons, further comprising the step of using melt signatures produced by said small double-stranded amplicons to distinguish between said at least two nearby neighbor variants. 
     
     
         72 . The method of  claim 70 , wherein melting of the small double-stranded amplicons and the probe/primer amplicons will provide thermal melt data for each type of amplicon. 
     
     
         73 . The method of  claim 71 , further comprising the step of analyzing melt data from both the small double-stranded amplicons and from the probe/primer amplicons to distinguish between said at least two nearby neighbor variants. 
     
     
         74 . A system for distinguishing between at least two nearby neighbor variants on a nucleic acid having a locus of interest comprising:
 (a) a microfluidic device comprising a plurality of sample loading zones, each of said sample loading zones being configured to house a separate asymmetric PCR using a nucleic acid having a locus of interest;   wherein said microfluidic device comprises at least two sample loading zones that are loaded with a limiting primer, an excess primer, and an unlabeled probe;   wherein at least one of said at least two sample loading zones that are loaded with an unlabeled probe contains a first unlabeled probe as the loaded unlabeled probe and at least one of said at least two sample loading zones that are loaded with an unlabeled probe contains a second unlabeled probe as the loaded unlabeled probe;   (b) a HRMA device, comprising a heating element, a fluorescence excitation light source and a fluorescence collection aperture, configured to thermally melt probe-amplicon elements obtained from asymmetric PCRs in said sample loading zones, and to generate fluorescence derivative melting curves for said probe-amplicon elements; and   (c) a fluorescence derivative melting curve analysis device configured to compare at least two melting curves generated by said HRMA device so as to distinguish between at least two nearby neighbor variants on the nucleic acid having a locus of interest.