US2001051363A1PendingUtilityA1
Methods for the elimination of DNA sequencing artifacts
Priority: Jan 30, 1995Filed: Jul 16, 2001Published: Dec 13, 2001
Est. expiryJan 30, 2015(expired)· nominal 20-yr term from priority
C12Q 1/6869C12Q 1/686
45
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Claims
Abstract
The present invention relates to improvements in methods of DNA sequencing. In particular, the invention relates to the elimination of stops or pauses in chain termination methods of DNA sequencing by the addition of nitrogen-containing organic compounds such as betaine, trimethylamine N-oxide and dimethylglycine. The invention also provides for DNA sequencing kits containing these compounds. The invention also provides for improvements in other laboratory procedures using DNA polymerases, such as polymerase chain reaction (PCR).
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method of decreasing the incidence of DNA polymerase stops occurring in a reaction mixture containing a DNA polymerase comprising adding to the reaction mixture an amount of a composition of the formula:
wherein:
R 1 , R 2 , and R 3 may be the same or different and are independently selected from the group consisting of hydrogen, methyl, ethyl, and propyl, with the proviso that no more than two of R 1 , R 2 , and R 3 are hydrogen; and
X is a moiety selected from the group consisting of:
radicals of the formulas
(a) ═O; and
(b)
wherein:
R 4 is selected from the group consisting of methyl and hydrogen and, when combined with R 1 , forms a pyrrolidine ring;
R 5 is selected from the group consisting of —O 2 H and —SO 3 H; and
n is an integer of from 0 to 2;
with the proviso that, when R 1 and R 4 form a pyrrolidine ring, no more than one of R 2 and R 3 is hydrogen, and wherein the composition is added in an amount effective to decrease the incidence of DNA polymerase stops.
2 . The method of claim 1 wherein R 1 , R 2 and R 3 are the same or different and selected from the group consisting of methyl, ethyl and hydrogen with the proviso that no more than two of R 1 , R 2 and R 3 are hydrogen and, when R 1 and R 4 form a pyrrolidine ring, no more than one of R 2 and R 3 is hydrogen.
3 . The method of claim 2 wherein X is —CH 2 CO 2 H.
4 . The method of claim 3 wherein R 1 , R 2 and R 3 are methyl.
5 . The method of claim 3 wherein R 1 , R 2 are methyl and R 3 is hydrogen.
6 . The method of claim 3 wherein R 1 is methyl and R 2 and R 3 are hydrogen.
7 . The method of claim 2 wherein X is ═O.
8 . The method of claim 7 wherein R 1 , R 2 and R 3 are methyl.
9 . The method of claim 2 wherein R 1 and R 4 form a pyrrolidine ring, R 2 and R 3 are methyl, n is 0, and R 5 is —CO 2 H.
10 . The method of claim 2 wherein R 1 , R 2 , and R 3 are methyl and X is —CH 2 SO 3 H.
11 . The method of claim 1 wherein said reaction mixture is a reaction mixture for a chain termination method of DNA sequencing.
12 . The method of claim 11 wherein said chain termination method of DNA sequencing is a dideoxy DNA sequencing method.
13 . The method of claim 1 wherein said reaction mixture is a PCR reaction mixture.
14 . The method of claim 1 wherein said reaction mixture comprises unpurified DNA.
15 . The method of claim 14 wherein said unpurified DNA is a crude cell lysate.
16 . A method of decreasing the incidence of DNA polymerase stops in a chain termination DNA sequencing method comprising the steps of:
a) combining in an aqueous solution, a DNA molecule; a DNA polymerase capable of producing a nucleic acid complementary to a portion of said DNA molecule by using the DNA molecule as a template; a mixture of deoxyribonucleoside triphosphates; a chain elongation inhibitor; and an amount of a composition of the formula: wherein: R 1 , R 2 , and R 3 may be the same or different and are independently selected from the group consisting of hydrogen, methyl, ethyl, and propyl, with the proviso that no more than two of R 1 , R 2 , and R 3 are hydrogen; and X is a moiety selected from the group consisting of: radicals of the formulas
(a) ═O; and
(b)
wherein:
R 4 is selected from the group consisting of methyl and hydrogen and, when combined with R 1 , forms a pyrrolidine ring; R 5 is selected from the group consisting of —CO 2 H and —SO 3 H; and n is an integer of from 0 to 2; with the proviso that, when R 1 and R 4 form a pyrrolidine ring, no more than one of R 2 and R 3 is hydrogen, and wherein the composition is added in an amount effective to decrease the incidence of DNA polymerase stops, to form a reaction mixture; and b) incubating the reaction mixture to permit the DNA polymerase to form nucleic acid fragments of varying length by using the DNA molecule as a template, wherein said nucleic acid fragments are complementary to said DNA molecule.
17 . The method of claim 16 wherein said chain elongation inhibitors are 2′,3′-dideoxyribonucleoside triphosphates.
18 . The method of claim 16 wherein R 1 , R 2 and R 3 are the same or different and selected from the group consisting of methyl, ethyl and hydrogen with the proviso that no more two of R 1 , R 2 and R 3 are hydrogen and, when R 1 and R 4 form a pyrrolidine ring, no more than one of R 2 and R 3 is hydrogen.
19 . The method of claim 18 wherein X is —CH 2 CO 2 H.
20 . The method of claim 19 wherein R 1 , R 2 and R 3 are methyl.
21 . The method of claim 19 wherein R 1 and R 2 are methyl and R 3 is hydrogen.
22 . The method of claim 19 wherein R 1 is methyl and R 2 and R 3 are hydrogen.
23 . The method of 18 wherein X is ═O.
24 . The method of claim 23 wherein R 1 , R 2 and R 3 are methyl.
25 . The method of claim 18 wherein R 1 and R 4 form a pyrrolidine ring, R 2 and R 3 are methyl, n is 0, and R 5 is —CO 2 H.
26 . The method of claim 18 wherein R 1 , R 2 , and R 3 are methyl and X is —CH 2 SO 3 H.
27 . The method of claim 16 wherein said DNA molecule is unpurified DNA.
28 . The method of claim 27 wherein said unpurified DNA is a crude cell lysate.
29 . The method of claim 16 wherein step a) further comprises the addition of a primer complementary to a second portion of said DNA molecule, said second portion of the DNA molecule located downstream to said first portion of the DNA molecule, wherein said DNA polymerase is capable of extending the 3′ end of said primer to produce a nucleic acid complementary to said first portion of the DNA molecule.
30 . A kit for sequencing DNA by a chain termination method comprising a container which contains one or more deoxyribonucleoside triphosphates, a container which contains a chain elongation inhibitor, and an amount of a composition of the formula:
wherein:
R 1 , R 2 , and R 3 may be the same or different and are independently selected from the group consisting of hydrogen, methyl, ethyl, and propyl, with the proviso that no more than two of R 1 , R 2 , and R 3 are hydrogen; and
X is a moiety selected from the group consisting of:
radicals of the formulas
(a) ═O; and
(b)
wherein:
R 4 is selected from the group consisting of methyl and hydrogen and, when combined with R 1 , forms a pyrrolidine ring;
R 5 is selected from the group consisting of —CO 2 H and —SO 3 H; and
n is an integer of from 0 to 2;
with the proviso that, when R 1 and R 4 form a pyrrolidine ring, no more than one of R 2 and R 3 is hydrogen.
31 . The kit of claim 30 wherein R 1 , R 2 and R 3 are the same or different and selected from the group consisting of methyl, ethyl and hydrogen with the proviso that no more two of R 1 , R 2 and R 3 are hydrogen and, when R 1 and R 4 form a pyrrolidine ring, no more than one of R 2 and R 3 is hydrogen.
32 . The kit of claim 31 wherein X is —CH 2 CO 2 H.
33 . The kit of claim 32 wherein R 1 and R 2 are methyl and R 3 is hydrogen.
34 . The kit of claim 32 wherein R 1 is methyl and R 2 and R 3 are hydrogen.
35 . The kit of claim 32 wherein R 1 , R 2 and R 3 are methyl.
36 . The kit of claim 31 wherein X is ═O.
37 . The kit of claim 36 wherein R 1 , R 2 and R 3 are methyl.
38 . The kit of claim 31 wherein R 1 and R 4 form a pyrrolidine ring, R 2 and R 3 are methyl, n is 0, and R 5 is —CO 2 H.
39 . The kit of claim 31 wherein R 1 , R 2 , and R 3 are methyl and X is —CH 2 —SO 3 H.
40 . The kit of claim 30 further comprising an amount of a DNA polymerase enzyme.Join the waitlist — get patent alerts
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