US2021285033A1PendingUtilityA1
Methods for variant detection
Est. expiryNov 25, 2035(~9.4 yrs left)· nominal 20-yr term from priority
Inventors:Caifu ChenJoseph DobosyPak Wah TsangMark Aaron BehlkeScott RoseKristin BeltzGarrett Richard Rettig
C12Q 2535/125C12Q 2525/186C12Q 2525/185C12Q 2525/161C12Q 2525/155C12Q 2525/121C12Q 2521/327C12N 9/1252C12N 2310/20C12N 9/22G16B 30/00C12Q 1/6827C12Y 301/26004C12Y 207/07007G16B 20/20C12N 15/11C12Q 1/6858C12Q 1/6853C12Q 2600/156C12Y 301/00C12Q 1/6876C07K 2319/21
74
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Claims
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.
Claims
exact text as granted — not AI-modified1 . 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
(
Δ
R
n
D
y
e
1
÷
Δ
Rn
D
y
e
2
)
×
1
2
0
9
0
;
and
(c) plotting on a circle plot with three axes, one for each dye or allele, the resulting distance.
13 . The method of claim 12 , the method comprising:
(a) using four fluorescent signals from multiple fluorescent dye signals in a single reaction well, subtracting a lowest fluorescence Dye 4 from fluorescence signals from Dye 1 , Dye 2 , and Dye 3 ; (b) calculating the distance of data from an origin and an angle from one of the axes with an equation:
Distance from origin=√{square root over ((Δ Rn Dye1 ) 2 +(Δ Rn Dye2 ) 2 )}
Angle=tan −1 (Δ Rn Dye 1 ÷ΔRn Dye 2 ); and
(c) plotting on a circle plot with four axes, one for each dye or allele, the resulting distance.
14 . The method of claim 12 , the method comprising:
(a) using five fluorescent signals from multiple fluorescent dye signals in a single reaction well, subtracting a lowest fluorescence Dye 5 from fluorescence signals from Dye 1 , Dye 2 , Dye 3 , and Dye 4 ; (b) calculating the distance of data from an origin and an angle from one of the axes 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
(
Δ
RnDye
1
÷
Δ
RnDye
2
)
×
72
9
0
;
and
(c) plotting on a circle plot with five axes, one for each dye or allele, the resulting distance.
15 . The method of claim 12 , the method comprising:
(a) using six fluorescent signals from multiple fluorescent dye signals in a single reaction well, subtracting a lowest fluorescence Dye 6 from fluorescence signals from Dye 1 , Dye 2 , Dye 3 , Dye 4 , and Dye 5 (b) calculating the distance of data from an origin and an angle from one of the axes 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
(
Δ
RnDye
1
÷
Δ
RnDye
2
)
×
60
9
0
;
and
(c) plotting on a circle plot with five axes, one for each dye or allele, the resulting distance.Cited by (0)
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