Methods for the elimination of DNA sequencing artifacts
AssigneeThe Regents of the University of California
InventorsMichael Chamberlin, Daniel Mytelka
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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).
Claims63
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:
- R1, R2, and R3 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 R1, R2, and R3 are hydrogen;
- and
- X is a moiety selected from the group consisting of:
- radicals of the formulas
- (a) ═O;
- and
- wherein:
- R4 is selected from the group consisting of methyl and hydrogen and, when combined with R1, forms a pyrrolidine ring;
- R5 is selected from the group consisting of —CO2H and —SO3H;
- and
- n is an integer of from 0 to 2;
- with the proviso that, when R1 and R4 form a pyrrolidine ring, no more than one of R2 and R3 is hydrogen, and wherein the composition is added in an amount effective to decrease the incidence of DNA polymerase stops.
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 deoxyribonucledside triphosphates;
- a chain elongation inhibitor;
- and an amount of a composition of the formula:
- wherein:
- R1, R2, and R3 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 R1, R2, and R3 are hydrogen;
- and
- X is a moiety selected from the group consisting of:
- radicals of the formulas
- (a) ═O;
- and
- wherein:
- R4 is selected from the group consisting of methyl and hydrogen and, when combined with R1, forms a pyrrolidine ring;
- R5 is selected from the group consisting of —CO2H and —SO3H;
- and
- n is an integer of from 0 to 2;
- with the proviso that, when R1 and R4 form a pyrrolidine ring, no more than one of R2 and R3 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.
The method of 18 wherein X is ═O.
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:
- R1, R2, and R3 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 R1, R2, and R3 are hydrogen;
- and
- X is a moiety selected from the group consisting of:
- radicals of the formulas
- (a) ═O;
- and
- wherein:
- R4 is selected from the group consisting of methyl and hydrogen and, when combined with R1, forms a pyrrolidine ring;
- R5 is selected from the group consisting of —CO2H and —SO3H;
- and
- n is an integer of from 0 to 2;
- with the proviso that, when R1 and R4 form a pyrrolidine ring, no more than one of R2 and R3 is hydrogen.
In the procedure for amplifying a target nucleotide sequence containing trinucleotide repeats, the improvement comprising adding an effective amount of a trimethylglycine to the reaction mixture of a Taq polymerase chain reaction procedure wherein fewer amplification products which do not correspond to the target nucleotide sequence are produced than would be produced in the absence of trimethylglycine.
A kit for amplifying a target nucleotide sequence containing trinucleotide repeats, comprising in separate containers:
- a) the components for a Taq polymerase chain reaction;
- and
- b) a trimethylglycine.
A kit for inhibiting stops during DNA polymerization comprising:
- a. a DNA polymerase;
- and
- b. a composition of the formula:
- wherein:
- R1, R2, and R3 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 R1, R2, and R3 are hydrogen;
- and
- X is a moiety selected from the group consisting of:
- radicals of the formulas
- (a) ═O;
- and
- wherein:
- R4 is selected from the group consisting of methyl and hydrogen and, when combined with R1, forms a pyrrolidine ring;
- R5 is selected from the group consisting of —CO2H and —SO3H;
- and
- n is an integer of from 0 to 2;
- with the proviso that, when R1 and R4 form a pyrrolidine ring, no more than one of R2 and R3 is hydrogen.
A method to reduce premature chain termination in the replication of a DNA molecule from a template DNA molecule, the method comprising the steps of:
- adding to a reaction mixture of the DNA template and a DNA polymerase an effective amount of chain termination reducing compound selected from the group consisting of trimethylglycine (betaine) and trimethylamine N-oxide (TMANO) so as to reduce premature chain termination which would otherwise occur in the absence of the chain termination reducing compound.
A method to reduce premature chain termination in the replication of a DNA molecule from a template DNA molecule, the method comprising the steps of:
- adding to a reaction mixture of the DNA template and a DNA polymerase an effective amount of trimethylglycine (betaine) so as to reduce premature chain termination which would otherwise occur in the absence of the trimethylglycine (betaine).
An improvement to methods for replication of a DNA molecule using a template DNA molecule, the improvement reducing premature chain termination, the improvement comprising the step of
- adding to a reaction mixture of the DNA template and a DNA polymerase an effective amount of a chain termination reducing compound selected from the group consisting of trimethylglycine (betaine) and trimethylamine N-oxide (TMANO) so as to reduce premature chain termination which would otherwise occur in the absence of the chain termination reducing compound.
An improvement to methods for replication of a DNA molecule using a template DNA molecule, the improvement reducing premature chain termination, the improvement comprising the step of
- adding to a reaction mixture of the DNA template and a DNA polymerase an effective amount of trimethylglycine (betaine) so as to reduce premature chain termination which would otherwise occur in the absence of the trimethylglycine (betaine).
In the procedure for replicating a template nucleotide sequence containing trinucleotide repeats inhibitory to chain elongation, the improvement comprising adding an effective amount of a trimethylglycine to the reaction mixture of a Taq polymerase DNA replication reaction procedure wherein fewer different replication products which do not correspond to the template nucleotide sequence are produced than would be produced in the absence of trimethylglycine.
A kit for replicating a target nucleotide sequence comprising:
- (a) a DNA polymerase;
- and
- (b) a trimethylglycine.
A kit for replicating a target nucleotide sequence containing trinucleotide repeats inhibitory to chain elongation, comprising:
- (a) the components for a Taq polymerase DNA replication procedure;
- and
- (b) a trimethylglycine.
In the procedure for sequencing a target nucleotide sequence containing trinucleotide repeats, the improvement comprising adding an effective amount of a trimethylglycine to the reaction mixture of a sequencing procedure wherein fewer sequencing products which do not correspond to the target nucleotide sequence are produced than would be produced in the absence of trimethylglycine.
Dependencies are inferred from the claim text.