Methods for variant detection

Patent metadata

Field	Value
Publication number	US-2017145486-A1
Application number	US-201615361280-A
Country	US
Kind code	A1
Filing date	Nov 25, 2016
Priority date	Nov 25, 2015
Publication date	May 25, 2017
Grant date	—

Abstract

The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays.

First claim

1 - 9 . (canceled)

10 : A method of detecting a variation in a target DNA sequence, the method comprising:
(a) providing a reaction mixture comprising:
(i) an oligonucleotide primer having a cleavage domain positioned 5′ of a blocking group and 3′ of a position of variation, the blocking group linked at or near the end of the 3′-end of the oligonucleotide primer wherein the blocking group prevents primer extension and/or inhibits the primer from serving as a template for DNA synthesis,
(ii) a sample nucleic acid that may or may not have the target sequence, and where the target sequence may or may not have the variation,
(iii) a cleaving enzyme, and
(iv) a polymerase;
(b) hybridizing the primer to the target DNA sequence to form a double-stranded substrate; (c) cleaving the hybridized primer, if the primer is complementary at the variation, with the cleaving enzyme at a point within or adjacent to the cleavage domain to remove the blocking group from the primer; and (d) extending the primer with the polymerase.

11 : The method of claim 10 , wherein the cleaving enzyme is a hot start cleaving enzyme that is thermostable and has reduced activity at lower temperatures.

12 : The method of claim 10 , wherein the cleaving enzyme is a chemically modified hot start cleaving enzyme that is thermostable and has reduced activity at lower temperatures.

13 : The method of claim 12 , wherein the hot start cleaving enzyme is a chemically modified Pyrococcus abyssi RNase H2.

14 : The method of claim 10 , wherein the cleaving enzyme is a hot start cleaving enzyme that is reversibly inactivated through interaction with an antibody at lower temperatures.

15 : The method of claim 10 , wherein the cleaving domain is comprised of at least one RNA base, and the cleaving enzyme cleaves between the position complementary to the variation and the RNA base.

16 : The method of claim 10 , wherein the cleaving domain is comprised of one or more 2′-modified nucleosides, and the cleaving enzyme cleaves between the position complementary to the variation and the one or more modified nucleosides.

17 : The method of claim 16 , wherein the one or more modified nucleosides are 2′-fluoronucleosides.

18 : The method of claim 10 , wherein the polymerase is a high-discrimination polymerase.

19 : The method of claim 10 , wherein the polymerase is a mutant H784Q Taq polymerase.

20 : The method of claim 19 , wherein the mutant H784Q Taq polymerase is reversibly inactivated via chemical, aptamer, or antibody modification.

21 : The method of claim 10 , wherein the primer contains a 5′ tail sequence that comprises a universal primer sequence and optionally a universal probe sequence, wherein the tail is non-complementary to the target DNA sequence.

22 - 53 . (canceled)

Assignees

• Integrated Dna Tech Inc

Inventors

• Chen Caifu
• Dobosy Joseph
• TSANG Pak Wah
• Behlke Mark Aaron
• Rose Scott
• Beltz Kristin
• RETTIG Garrett

Classifications

• C12Q1/6827 Primary

  for detection of mutation or polymorphism · CPC title

• C12Q2535/125

  Allele specific primer extension · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q2525/185

  incorporating bases where the precise position of the bases in the nucleic acid string is important · CPC title

• C12Q2525/161

  incorporating target specific and non-target specific sites · CPC title