Methods for variant detection

Patent metadata

Field	Value
Publication number	US-2021285033-A1
Application number	US-202017084797-A
Country	US
Kind code	A1
Filing date	Oct 30, 2020
Priority date	Nov 25, 2015
Publication date	Sep 16, 2021
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 . A method of detecting one or more variations in a target DNA sequence, the method comprising:
(a) providing a first reaction mixture comprising:
(i) a first allele specific oligonucleotide primer and a second allele specific oligonucleotide primer, both 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 first and second allele specific oligonucleotide primers from serving as a template for DNA synthesis,
(ii) a nucleic acid sample that may or may not have the target DNA sequence, wherein the target DNA sequence may or may not have the variation,
(iii) a cleaving enzyme, and
(iv) a polymerase, wherein the polymerase is a high-discrimination mutant H784Q Taq polymerase;
(b) hybridizing the first and second allele specific oligonucleotide primers to the target DNA sequence, if present in the sample, to form a double-stranded substrate; (c) cleaving the first and second allele specific oligonucleotide primers hybridized to the target DNA sequence, if the first and second allele specific oligonucleotide primers are 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 first and second allele specific oligonucleotide primers; and (d) extending the first and second allele specific oligonucleotide primers with the high-discrimination mutant H784Q Taq polymerase; and (e) providing a second reaction mixture comprising:
(i) the first allele specific oligonucleotide primer and a third allele specific oligonucleotide primer, both 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 first and second allele specific oligonucleotide primers, wherein the blocking group prevents primer extension and/or inhibits the first and third allele specific oligonucleotide primers from serving as a template for DNA synthesis,
(ii) a nucleic acid sample that may or may not have the target DNA sequence, wherein the target DNA sequence may or may not have the variation,
(iii) a cleaving enzyme, and
(iv) a polymerase, wherein the polymerase is a high-discrimination mutant H784Q Taq polymerase;
(f) hybridizing the first and third allele specific oligonucleotide primers to the target DNA sequence, if present in the sample, to form a double-stranded substrate; (g) cleaving the first and third oligonucleotide primers hybridized to the target DNA sequence, if the first and third allele specific oligonucleotide primers are 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 first and third allele specific oligonucleotide primers; and (h) extending the first and third allele specific oligonucleotide primers with the high-discrimination mutant H784Q Taq polymerase.

2 . The method of claim 1 , wherein the first, second, and third allele specific oligonucleotide primers contain:
(a) a 5′ tail sequence that comprises a universal primer sequence and a reporter probe sequence that corresponds to an allele specific oligonucleotide primer, wherein the 5′ tail sequence is non-complementary to the target DNA sequence; (b) a region complementary to the target DNA sequence; and (c) an allele specific domain.

3 . The method of claim 2 , wherein the allele specific domain is capable of being cleaved by an RNase H enzyme when hybridized to the target DNA sequence.

4 . The method of claim 1 , wherein the cleavage 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.

5 . The method of claim 1 , wherein the cleavage 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 2′-modified nucleosides.

6 . The method of claim 5 , wherein the one or more 2′-modified nucleosides are 2′-fluoronucleosides.

7 . The method of claim 1 , wherein the cleaving enzyme is a hot start cleaving enzyme that is reversibly inactivated through interaction with an antibody at lower temperatures.

8 . The method of claim 1 , wherein the cleaving enzyme is a hot start cleaving enzyme that is thermostable and has reduced activity at lower temperatures.

9 . The method of claim 1 , wherein the cleaving enzyme is a chemically modified hot start cleaving enzyme that is thermostable and has reduced activity at lower temperatures.

10 . The method of claim 9 , wherein the chemically modified hot start cleaving enzyme is a chemically modified Pyrococcus abyssi RNase H2.

11 . The method of claim 1 , wherein the high-discrimination mutant H784Q Taq polymerase is reversibly inactivated via chemical, aptamer, or antibody modification.

12 . A method of visualization of multiple different fluorescent signals from allelic amplification plots, the method comprising:
(a) using three fluorescent signals from multiple fluorescent dye signals in a single reaction well, subtracting a lowest fluorescence Dye 3 from fluorescence signals from Dye 1 and Dye 2 ; (b) calculating the distance of data from an origin and an angle from one of the axis with an equation

Distance
⁢

⁢
from
⁢

⁢
origin

=

(

Δ
⁢
R
⁢

n

D
⁢
y
⁢
e
⁢
1

)

2

+

(

Δ
⁢
R
⁢

n

D
⁢
y
⁢
e
⁢
2

)

2

Angle
=

tan

-
1

⁡

(

Assignees

• Integrated Dna Tech Inc

Inventors

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

Classifications

• 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

• C12Q2525/155

  incorporating/generating a new priming site · CPC title