초록 ▼

Provided herein are methods, compositions, and kits for detecting alleles using a single probe or a single primer set. Also, provided herein are methods, compositions, and kits for detecting allelic variants using a single probe or a single primer set. Also, provided herein are methods, compositions

Provided herein are methods, compositions, and kits for detecting alleles using a single probe or a single primer set. Also, provided herein are methods, compositions, and kits for detecting allelic variants using a single probe or a single primer set. Also, provided herein are methods, compositions, and kits for determining a polymerization error rate.

대표청구항 ▼

1. A method for detecting a first and a second allele of a target locus in a plurality of target polynucleotide molecules, comprising: (a) partitioning a sample comprising said target polynucleotide molecules into a plurality of fluid reaction volumes;(b) performing an amplification reaction in said

1. A method for detecting a first and a second allele of a target locus in a plurality of target polynucleotide molecules, comprising: (a) partitioning a sample comprising said target polynucleotide molecules into a plurality of fluid reaction volumes;(b) performing an amplification reaction in said reaction volumes, wherein each reaction volume comprises (i) a forward primer that is complementary to a first sequence of a first strand of the target polynucleotide molecules, wherein the first sequence is 5′ of the target locus, (ii) a reverse primer that is complementary to a second sequence of a second strand of the target polynucleotide molecules, wherein the second sequence is 3′ of the target locus, and (iii) a detection probe that is capable of hybridizing to a third sequence of the target polynucleotide molecules that encompasses the target locus, wherein the detection probe comprises a first signal reporter; and(c) determining a number and/or fraction of positive reaction volumes containing a target molecule having the first allele and a number and/or fraction of reaction volumes containing a target molecule having the second allele based on a signal of the first signal reporter detected from the reaction volumes. 2. A method for detecting a first allele and a second allele of a target locus in a plurality of target polynucleotide molecules, comprising: (a) partitioning a sample comprising said target polynucleotide molecules into a plurality of fluid reaction volumes;(b) performing an amplification reaction in said reaction volumes, wherein each reaction volume comprises (i) a forward primer that is complementary to a first sequence of a first strand of the target polynucleotide molecules, (ii) a reverse primer that is complementary to a second sequence of a second strand of the target polynucleotide molecules, wherein the target locus resides within the first sequence or the second sequence, and (iii) a detection probe that (A) is capable of hybridizing to a third sequence of the target polynucleotide molecules, wherein the third sequence is located between the first sequence and the second sequence, and wherein the detection probe comprises a first signal reporter, or (B) comprises a first signal reporter that produces a more intense signal when bound to DNA than when not bound to DNA, wherein amplification is performed optionally using a nucleic acid polymerase; and(c) determining a number and/or fraction of positive reaction volumes comprising a target molecule having the first allele and a number and/or fraction of reaction volumes containing a target molecule having the second allele based on a signal of the first signal reporter detected from the reaction volumes. 3. A method for determining a polymerase error rate comprising: (a) performing a first amplification of a sample comprising a plurality of target polynucleotide molecules, wherein a target polynucleotide comprises a target locus comprising a restriction enzyme recognition site, and thereby generating a plurality of amplicons;(b) providing an oligonucleotide that is complementary to a target sequence of the target polynucleotide molecules, wherein the target sequence comprises the target locus;(c) digesting the amplicons into a digested sample;(d) partitioning said digested sample into a plurality of fluid reaction volumes;(e) performing an amplification reaction in the reaction volumes, wherein each reaction volume comprises (i) a forward primer that is complementary to a first sequence of a first strand of the target polynucleotide molecules, wherein the first sequence is 5′ of the target locus, (ii) a reverse primer that is complementary to a second sequence of a second strand of the target polynucleotide molecules, wherein the second sequence is 3′ of the target locus, (iii) a detection probe that is capable of hybridizing to the target sequence, wherein the detection probe comprises a first signal reporter, and (iv) a reference probe that is capable of hybridizing to a third sequence of the target polynucleotide molecules located between the first sequence and the second sequence that encompasses a reference locus, wherein the reference probe comprises a second signal reporter;(f) determining a number and/or fraction of positive reaction volumes containing a target molecule comprising a mutation in the restriction enzyme recognition site based on a signal of the first signal reporter detected from the reaction volumes; and(g) determining a number and/or fraction of positive reaction volumes containing a target molecule comprising a mutation in the reference locus based on a signal of the second signal reporter detected from the reaction volumes. 4. The method of claim 1, wherein the degree of signal corresponds to the degree of nucleic acid amplification. 5. The method of claim 1, wherein the number and/or fraction of positive reaction volumes is used to quantify the target molecule having the first allele and/or the target molecule having the second allele. 6. The method of claim 1, wherein the degree of signal corresponds to the degree of matching between the detection probe and the target locus. 7. The method of claim 1, wherein the reaction volumes are droplets. 8. The method of claim 1, wherein the reaction volumes are contained in chambers. 9. The method of claim 1, wherein the reaction volumes are between about 1 pL and about 100 mL. 10. The method of claim 1, further comprising determining the concentration of nucleic acids in the sample and/or reaction volumes. 11. The method of claim 1, wherein the concentration of nucleic acids in the sample and/or reaction volumes is known. 12. The method of claim 1, wherein more bases of the detection probe anneal to the first allele of the target locus than to the second allele of the target locus. 13. The method of claim 1, wherein the detection probe comprises at least one base that is not complementary to the second allele of the target locus. 14. The method of claim 1, wherein the reaction volumes do not contain an enzyme comprising endonuclease activity. 15. The method of claim 1, further comprising detecting a gene copy number alteration associated with a disease. 16. The method of claim 15, further comprising detecting a mutation within a gene that has the copy number alteration. 17. The method of claim 1, wherein the reaction volumes further comprise a reference probe capable of hybridizing to a fourth sequence of the target polynucleotide molecules, wherein the fourth sequence is located between the first sequence and the second sequence, wherein the reference probe hybridizes to a reference locus that does not contain a variant, and wherein the reference probe comprises a second signal reporter. 18. The method of claim 1, wherein the reaction volumes further comprise (i) a reference forward primer that is complementary to a fourth sequence of the target polynucleotide molecules, wherein the fourth sequence is 5′ of a reference locus that does not contain a variant; (ii) a reference reverse primer that is complementary to a fifth sequence of the target polynucleotide molecules, wherein the fifth sequence is 3′ of the reference locus; and (iii) a reference probe capable of hybridizing to the reference locus, wherein the reference probe comprises a second signal reporter. 19. The method of claim 17, wherein the first and fourth sequences are located on the same target polynucleotide. 20. The method of claim 17, wherein the first and fourth sequences are not located on the same target polynucleotide. 21. The method of claim 17, wherein the first and fourth sequences are located on different chromosomes. 22. The method of claim 17, wherein the amplification reaction is performed using a nucleic acid polymerase having 5′-3′ nuclease activity that digests the reference probe during amplification to separate the second signal reporter from a quencher. 23. The method of claim 17, wherein the amplification reaction is performed using a nucleic acid polymerase, and wherein the reference probe fluoresces more when annealed to amplified target than when not annealed to an amplified target. 24. The method of claim 17, further comprising determining a number of reaction volumes containing a reference target molecule based on a signal of the second signal reporter detected from each of the reaction volumes. 25. The method of claim 17, further comprising determining the presence or absence of a reference target molecule based on a signal of the second signal reporter detected from the reaction volumes. 26. The method of claim 17, wherein a signal detected from the second signal reporter corresponds to the occurrence of reference nucleic acid amplification. 27. The method of claim 17, wherein the reference locus is used as a reference to measure the number of genome equivalents in the reaction volumes. 28. The method of claim 17, wherein the reference locus is used as a reference to compare the concentration of the target locus to the reference locus and can be used to determine amplification or deletion of a target locus. 29. The method of claim 17, wherein the reference probe is a nonspecific detection probe. 30. The method of claim 17, further comprising determining the concentration of the target nucleotide and the reference target nucleotide. 31. The method of claim 17, further comprising (d) digesting the sample comprising a plurality of target polynucleotide molecules with a restriction enzyme before performing the amplification reaction, wherein the third sequence comprises a restriction enzyme recognition site of the restriction enzyme. 32. The method of claim 1, wherein the reaction volumes further comprise (i) a reference forward primer that is complementary to a fourth sequence of the target polynucleotide molecules, wherein the fourth sequence is 5′ of a reference locus, (ii) a reference reverse primer that is complementary to a fifth sequence of the target polynucleotide molecules, wherein the fifth sequence is 3′ of the reference locus, and (iii) a reference probe that is a nonspecific detection probe. 33. The method of claim 1, wherein the reaction volumes further comprise a reference probe that is a nonspecific detection probe. 34. The method of claim 33, wherein the nonspecific detection probe includes a dye that binds to double stranded nucleic acid. 35. The method of claim 34, wherein multiple copies of the dye bind to double-stranded nucleic acid in direct relation to a length of the nucleic acid. 36. The method of claim 34, wherein the dye is an intercalating dye. 37. The method of claim 34, wherein the dye becomes more luminescent when bound to the double-stranded nucleic acid. 38. The method of claim 1, wherein at least one of the reaction volumes comprises both the first allele and the second allele. 39. The method of claim 17 or 34, further comprising determining an amplified or deleted allele. 40. The method of claim 1, further comprising genotyping the sample. 41. The method of claim 1, wherein the first signal reporter is a fluorescence reporter and the signal is fluorescence. 42. The method of claim 1, wherein the detection probe further comprises a first quencher. 43. The method of claim 42, wherein in the presence of the target locus at the annealing temperature the first signal reporter and the first quencher are spatially separated, and in the absence of the target locus the first signal reporter is quenched. 44. The method of claim 42, wherein the amplification reaction is performed (A) using a nucleic acid polymerase having 5′-3′ nuclease activity that digests the detection probe during amplification to separate the first signal reporter from the first quencher, or (B) using a nucleic acid polymerase and the detection probe anneals to amplified target, or (C) by isothermal DNA amplification. 45. The method of claim 17, wherein the second signal reporter is a fluorescence reporter. 46. The method of claim 17, wherein the reference probe further comprises a second quencher. 47. The method of claim 46, wherein in the presence of the reference locus at the annealing temperature the second signal reporter and the second quencher are spatially separated, and in the absence of target locus the second signal reporter is quenched. 48. The method of claim 38, wherein at least one of the reaction volumes comprises no target polynucleotide molecules.