IPC분류정보
국가/구분 |
United States(US) Patent
공개
|
국제특허분류(IPC7판) |
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출원번호 |
16301202
(2017-05-18)
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공개번호 |
20190144950
(2019-05-16)
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국제출원번호 |
PCT/US17/33314
(2017-05-18)
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발명자
/ 주소 |
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출원인 / 주소 |
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인용정보 |
피인용 횟수 :
0 인용 특허 :
0 |
초록
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The present disclosure is directed to digital PCR (dPCR)-based methods and kits for detecting and quantifying mutant nucleic acids (e.g., transcripts) of a gene containing insertions at specific locations. In some embodiments, the method permits sensitive detection and quantitation of mutant NPM1 nu
The present disclosure is directed to digital PCR (dPCR)-based methods and kits for detecting and quantifying mutant nucleic acids (e.g., transcripts) of a gene containing insertions at specific locations. In some embodiments, the method permits sensitive detection and quantitation of mutant NPM1 nucleic acids comprising insertion mutations (NPM1mut subtypes).
대표청구항
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1. A method of detecting mutant nucleic acid molecules of a gene in a sample, comprising: providing a sample suspected of containing mutant nucleic acid molecules of said gene;performing digital PCR on said sample using a first primer set, a second primer, and a first probe, wherein(i) the first pri
1. A method of detecting mutant nucleic acid molecules of a gene in a sample, comprising: providing a sample suspected of containing mutant nucleic acid molecules of said gene;performing digital PCR on said sample using a first primer set, a second primer, and a first probe, wherein(i) the first primer set comprises multiple primers, wherein the multiple primers anneal specifically to mutant nucleic acid molecules of the gene that each comprise an insertion of at least one nucleotide at a specific site in the gene, and the multiple primers are degenerate in at least one nucleotide position corresponding to the position of an inserted nucleotide in the mutant nucleic acid molecules;(ii) the second primer anneals specifically to a sequence in said gene to permit generation of amplicons when used with the first primer set in the digital PCR, and(iii) the first probe hybridizes to a sequence in said gene located between the site of insertion and the sequence to which the second primer hybridizes. 2. The method of claim 1, wherein the multiple primers in the first primer set are degenerate in 2-4 nucleotide positions. 3. The method of claim 1, wherein said multiple primers in the first primer set comprises nucleotides at four adjacent positions (NNNN) corresponding to positions of inserted nucleotides in the mutant nucleic acid molecules, wherein N at each position is independently A, C, G, T, dI or 5NI, and wherein the multiple primers are degenerate at at least two of the four positions. 4. The method of claim 3, wherein the multiple primers are degenerate at all four positions. 5. The method according to claim 1, wherein degeneracy at a nucleotide position in the first primer set is achieved by using a mixture of selected nucleotides at a predetermined ratio. 6. The method of claim 5, wherein the mixture of selected nucleotides are a mixture of A, C, G, and T, a mixture of C and T, a mixture of A and T, or a mixture of A and G. 7. The method of according to claim 1, wherein the first primer set and the second primer are designed to provide amplicons of about 75-200 base pairs. 8. The method of according to claim 1, wherein the gene is NPM1. 9. The method of claim 8, wherein the specific site of insertion is between positions 863 and 864, positions 860 and 861, positions 861 and 862, and positions 867 and 868, of the wild-type NPM1 coding sequence. 10. The method of claim 9, wherein the specific site of insertion is between positions 863 and 864, and wherein when said second primer is a forward primer, said second primer hybridizes to a sequence in exon 9, 10, 11 or a combination of exons 9, 10 and 11 of NPM1, and when said second primer is a reverse primer, said second primer hybridizes to a sequence in exon 12 of NPM1. 11. The method of claim 8, wherein when said multiple primers in the first primer set are forward primers, said second primer is a reverse primer and each primer in the first primer set comprises the sequence, 5′-TCTGNNNNGCAGTGGAGGAAG-3′ (SEQ ID NO: 12), wherein N at each position is independently A, C, T, G, dI or 5NI, and at least one of the four positions denoted by N is degenerate; and wherein when said multiple primers in the first primer set are reverse primers, said second primer is a forward primer and each primer in the first primer set comprises the sequence, 5′-CTTCCTCCACTGCNNNNCAGA-3′ (SEQ ID NO: 4), wherein N at each position is independently A, C, T, G, dI or 5NI, and at least one of the four positions denoted by N is degenerate. 12. The method of claim 11, wherein at least one N is fixed to a nucleotide selected from the group consisting of A, T, G or C. 13. The method of claim 12, wherein said multiple primers in the first primer set are forward primers represented by the sequence to 5′-TCTGYWTGGCAGTGGAGGAAG-3′ (SEQ ID NO: 13), wherein Y is C or T and W is A or T; or wherein said multiple primers in the first primer set are reverse primers represented by the sequence 5′-CTTCCTCCACTGCCAWRCAGA-3′ (SEQ ID NO: 14), wherein W is A or T and R is A or G. 14. The method of claim 10, wherein the second primer comprises the sequence, 5′-GAAGAATTGCTTCCGGATGACT-3′ (SEQ ID NO: 1). 15. The method of claim 10, wherein the first probe comprises the sequence, 5′-ACCAAGAGGCTATTCAA-3′ (SEQ ID NO: 2). 16. The method of claim 1, wherein the first probe comprises a fluorescent label and a quencher. 17. The method of claim 1, wherein the mutant nucleic acids are RNA, cDNA or DNA. 18. The method of claim 10, further comprising quantifying the mutated nucleic acids of the NPM1 gene in the sample. 19. The method of claim 18, further comprising quantifying nucleic acids of a second gene in the sample and generating a ratio of mutant nucleic acids of the NPM1 gene to the nucleic acids of the second gene. 20. The method of claim 19, wherein the second gene is ABL1, wild-type NPM1, or total NPM1. 21. The method of claim 20, wherein a second probe that specifically hybridizes to ABL1 is used to quantify ABL1, and wherein the second probe comprises a second fluorescent label different from the fluorescent label of the first probe. 22. The method according to claim 18, wherein the number of any mutated NPM1 transcript greater than or equal to a detection limit is indicative of cancer or residual cancer cells. 23. The method of claim 22, where the cancer is a blood cancer. 24. The method of claim 23, wherein the blood cancer is selected from the group consisting of non-Hodgkin's lymphoma, acute promyelocytic leukemia, myelodysplastic syndrome, acute lymphocytic leukemia and acute myelogenous leukemia. 25. The method of claim 1, wherein the sample is a sample of a cancer patient who has undergone cancer therapy. 26. A kit comprising a first primer set which comprises multiple primers, wherein said multiple primers specifically anneal to mutant NPM1 nucleic acid molecules encompassing an insertion of at least one nucleotide at a specific site in NPM1, and wherein said multiple primers are degenerate in at least one position corresponding to a position of an inserted nucleotide in the mutant NPM1 nucleic acid molecules. 27. The kit of claim 26, wherein said specific site is between positions 859 and 860, positions 860 and 861, positions 861 and 862, positions 862 and 863, positions 863 and 864, positions 864 and 865, positions 865 and 866, positions 866 and 867, and positions 867 and 868, of the wild-type NPM1 coding sequence. 28. The kit of claim 26, wherein said multiple primers within the first primer set are degenerate in at least one of four adjacent positions (5′ NNNN 3′) corresponding to positions of inserted nucleotides in the mutant NPM1 nucleic acid molecules, and wherein N at each position is independently A, C, G or T. 29. The kit of claim 28, wherein at least one N is fixed to a nucleotide selected from the group consisting of A, T, G or C. 30. The kit of claim 29, wherein the first N is Y, the second N is W, the third N is T and the fourth N is G, wherein Y is C or T and W is A or T 31. The kit of claim 30, wherein said multiple primers in the first primer set are forward primers represented by the sequence to 5′-TCTGYWTGGCAGTGGAGGAAG-3′ (SEQ ID NO: 13), or said multiple primers in the first primer set are reverse primers represented by the sequence to 5′-CTTCCTCCACTGCCAWRCAGA-3′ (SEQ ID NO: 14), wherein W is A or T and R is A or G. 32. The kit of claim 26, further comprising a second primer that hybridizes to a sequence in NPM1, wherein the first primer set and the second primer are designed to provide amplicons of about 75-200 base pairs in length. 33. The kit of claim 32, further comprising a probe hybridizes to a sequence in NPM1 located between the site of insertion in NPM1 and the sequence in NPM1 to which the second primer hybridizes. 34. The kit of claim 33, wherein said probe is labeled with a fluorophore and a quencher.
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