Methods for rapid and sensitive detection of hotspot mutations

Patent metadata

Field	Value
Publication number	US-10633711-B2
Application number	US-201515505777-A
Country	US
Kind code	B2
Filing date	Aug 24, 2015
Priority date	Aug 25, 2014
Publication date	Apr 28, 2020
Grant date	Apr 28, 2020

Abstract

Methods that rapidly, sensitively, and specifically detect mutations in IDH1/2 and the TERT promoter employ amplification of particular portions of the genes that experience frequent and exquisitely localized mutations. The ability to distinguish between sequences that differ only by one nucleotide and which may be present in very low ratios is essential for such an assay.

First claim

We claim:

1. A method of testing a body sample of a human with a tumor, comprising:
amplifying tumor DNA of a body sample of the human with a set of allele-specific amplification primers, wherein the set of allele-specific primers comprises sequences of SEQ ID NO: 2, 5, 8, and 12, wherein said allele-specific primers comprise an LNA-modified nucleotide at their 3′ ends, wherein said amplifying further employs allele-non-specific amplification primers, wherein said allele-non-specific primers comprise sequences SEQ ID NO: 3, 6, 9, 10, 13, and 14 to generate amplification products comprising TERT promoter and IDH1 and IDH2 sequences when complementary templates are present in the body sample; and
detecting the amplification products of said tumor DNA, wherein 0.1% mutant genomic DNA copies in a background of wild-type copies can be detected, and wherein 0.1% mutant genomic copies can be distinguished from 0% mutant genomic copies.

2. A method of testing a body sample of a human with a tumor, comprising:
amplifying tumor DNA of a body sample of the human with a set of primers comprising the sequences of SEQ ID NO: 1-14, wherein the set comprises allele-specific amplification primers which comprise sequences of SEQ ID NO: 2, 5, 8, and 12, wherein said allele-specific primers comprise an LNA-modified nucleotide at their 3′ ends, wherein said set further comprises allele-non-specific amplification primers which comprise sequences of SEQ ID NOS: 3, 6, 9, 10, 13, and 14, to generate amplification products comprising TERT promoter and IDH1 and IDH2 sequences when complementary templates are present in the body sample; and
detecting the amplification products of said tumor DNA, wherein 0.1% mutant genomic DNA copies in a background of wild-type copies can be detected, and wherein 0.1% mutant genomic copies can be distinguished from 0% mutant genomic copies.

3. The method of claim 1 or 2 wherein the set of allele-specific amplification primers further comprises sequences of SEQ ID NO: 15-21.

4. The method of claim 3 wherein a tag sequence is attached to the 5′ end of at least one of the primers.

5. The method of claim 1 or 2 wherein the tumor DNA is genomic DNA.

6. The method of claim 1 or 2 wherein the tumor DNA is genomic DNA that has been subjected to pre-amplification.

7. The method of claim 6 wherein the pre-amplification employs a DNA polymerase having a higher fidelity rate than Taq polymerase.

8. The method of claim 6 wherein the pre-amplification employs an annealing temperature of ≥66° C.

9. The method of claim 6 wherein the pre-amplification employs an annealing temperature of ≥68° C.

10. The method of claim 6 wherein the pre-amplification is conducted as a multiplex reaction.

11. The method of claim 1 or 2 wherein at least a portion of cycles of the amplifying are conducted at ≥66° C.

12. The method of claim 11 wherein a portion of the cycles of the amplifying are conducted at ≤60° C.

13. The method of claim 2 wherein separate aliquots of the tumor DNA of the body sample are amplified with sets of primers comprising (a) SEQ ID NO: 1-3; (b) SEQ ID NO: 4-6; (c) SEQ ID NO: 3 and 6; (d) SEQ ID NO: 7-9; (e) SEQ ID NO: 9 and 10; (f) SEQ ID NO: 11-13; and (g) SEQ ID NO: 13-14.

14. The method of claim 1 or 2 wherein the amplification is performed as a quantitative PCR.

15. The method of claim 1 or 2 wherein the body sample is selected from the group consisting of cerebral spinal fluid (CSF), blood, lymph, serum, plasma, urine, saliva, mucus, and tears.

16. The method of claim 1 or 2 wherein the body sample is a biopsy sample.

17. The method of claim 1 or 2 wherein the body sample is a needle aspirate.

18. The method of claim 1 or 2 wherein a tag sequence is attached to the 5′ end of at least one of the primers.

19. The method of claim 1 wherein said set of allele-specific primers comprise primers each of which consist of a sequence selected from the group consisting of SEQ ID NO: 2, 5, 8 and 12.

20. The method of claim 1 wherein said set further comprises primers each of which consists of a sequence of SEQ ID NO: 1, 4, 7, and 11.

21. The method of claim 2 wherein said allele-specific primers comprise primers each of which consists of a sequence selected from the group consisting of SEQ ID NO: 1, 2, 4, 5, 7, 8, 11, and 12.

Assignees

• Univ Duke

Inventors

• Yan Hai
• He Yiping
• Yang Rui
• Diplas Bill H
• Hansen Landon
• Bigner Darell

Classifications

• C12Q2600/16

  Primer sets for multiplex assays · CPC title

• C12Q1/6886 Primary

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q2600/166

  Oligonucleotides used as internal standards, controls or normalisation probes · CPC title

• C12Q2527/101

  Temperature · CPC title