Compositions, methods, and kits for analyzing DNA methylation
AssigneeApplera Corporation
InventorVictoria Boyd
Life of this patent
Estimating the term…
Abstract
Compositions, methods, and kits for reducing strand amplification bias using bisulfite treated gDNA are provided. Methods for detecting and for quantitating the amplified bisulfite treated gDNA and inferring the presence, absence, and/or degree of methylation of target cytosine(s) in the gDNA are also provided. Such methods typically employ tailed first primer pairs, which can, but need not comprise nucleotide analogs, and optionally second primer pairs.
Claims69
A method for reducing strand amplification bias with bisulfite treated genomic DNA (gDNA) comprising:
- (a) annealing a first primer with the bisulfite treated gDNA at a first annealing temperature, wherein the first primer comprises:
- (i) a target-complementary portion and (ii) a first primer-binding site upstream from the target-complementary portion;
- (b) extending the annealed first primer to generate a first extension product;
- (c) annealing a second primer with the first extension product at a second annealing temperature, wherein the second primer comprises:
- (i) a first extension product-complementary portion and (ii) a second primer-binding site upstream from the first extension product-complementary portion;
- (d) extending the annealed second primer to generate a second extension product;
- (e) annealing an additional first primer with the second extension product at a third annealing temperature;
- (f) extending the annealed first primer to generate a third extension product;
- (g) annealing an additional second primer with the third extension product at a fourth annealing temperature;
- (h) extending the annealed second primer to generate an additional second extension product;
- and
- (i) optionally, repeating steps (e)-(h) at least one additional cycle.
A method for reducing strand amplification bias with bisulfite treated gDNA comprising:
- (a) annealing a first primer with the bisulfite treated gDNA at a first annealing temperature, wherein the first primer comprises:
- (i) a target-complementary portion comprising a nucleotide analog and (ii) a first primer-binding site upstream from the target-complementary portion;
- (b) extending the annealed first primer to generate a first extension product;
- (c) annealing a second primer with the first extension product at a second annealing temperature, wherein the second primer comprises:
- (i) a first extension product-complementary portion comprising a nucleotide analog and (ii) a second primer-binding site upstream from the first extension product-complementary portion;
- (d) extending the annealed second primer to generate a second extension product;
- (e) annealing an additional first primer with the second extension product at a third annealing temperature;
- (f) extending the annealed first primer to generate a third extension product;
- (g) annealing an additional second primer with the third extension product at a fourth annealing temperature;
- (h) extending the annealed second primer to generate an additional second extension product;
- and
- (i) optionally, repeating steps (e)-(h) at least one additional cycle.
A method for reducing strand amplification bias with bisulfite treated gDNA comprising:
- (a) annealing a first primer with the bisulfite treated gDNA at a first annealing temperature, wherein the first primer comprises:
- (i) a target-complementary portion and (ii) a first primer-binding site upstream from the target-complementary portion;
- (b) extending the annealed first primer to generate a first extension product comprising a nucleotide analog;
- (c) annealing a second primer with the first extension product at a second annealing temperature, wherein the second primer comprises:
- (i) a first extension product-complementary portion and (ii) a second primer-binding site upstream from the first extension product-complementary portion;
- (d) extending the annealed second primer to generate a second extension product comprising a nucleotide analog;
- (e) annealing an additional first primer with the second extension product at a third annealing temperature;
- (f) extending the annealed first primer to generate a third extension product;
- (g) annealing an additional second primer with the third extension product at a fourth annealing temperature;
- (h) extending the annealed second primer to generate an additional second extension product;
- and
- (i) optionally, repeating steps (e)-(h) at least one additional cycle.
A method for reducing strand amplification bias with bisulfite treated gDNA comprising:
- (a) annealing a first primer with the bisulfite treated gDNA at a first annealing temperature, wherein the first primer comprises:
- (i) a target-complementary portion and (ii) a first primer-binding site upstream from the target-complementary portion;
- (b) extending the annealed first primer to generate a first extension product;
- (c) annealing a second primer with the first extension product at a second annealing temperature, wherein the second primer comprises:
- (i) a first extension product-complementary portion and (ii) a second primer-binding site upstream from the first extension product-complementary portion;
- (d) extending the annealed second primer to generate a second extension product;
- (e) annealing a third primer with the complement of the first primer-binding site of the second extension product at a third annealing temperature;
- (f) extending the annealed third primer to generate a third extension product;
- (g) annealing a fourth primer with the complement of the second primer-binding site of the third extension product at a fourth annealing temperature;
- (h) extending the annealed fourth primer to generate an additional second extension product;
- and
- (i) optionally, repeating steps (e)-(h) at least one additional cycle.
A method for reducing strand amplification bias with bisulfite treated gDNA comprising:
- (a) annealing a first primer with the bisulfite treated gDNA at a first annealing temperature, wherein the first primer comprises:
- (i) a target-complementary portion comprising a nucleotide analog and (ii) a first primer-binding site upstream from the target-complementary portion;
- (b) extending the annealed first primer to generate a first extension product;
- (c) annealing a second primer with the first extension product at a second annealing temperature, wherein the second primer comprises:
- (i) a first extension product-complementary portion comprising a nucleotide analog and (ii) a second primer-binding site upstream from the first extension product-complementary portion;
- (d) extending the annealed second primer to generate a second extension product;
- (e) annealing a third primer with the complement of the first primer-binding site of the second extension product at a third annealing temperature;
- (f) extending the annealed third primer to generate a third extension product;
- (g) annealing a fourth primer with the complement of the second primer-binding site of the third extension product at a fourth annealing temperature;
- (h) extending the annealed fourth primer to generate an additional second extension product;
- and
- (i) optionally, repeating steps (e)-(h) at least one additional cycle.
A method for reducing strand amplification bias with bisulfite treated gDNA comprising:
- (a) annealing a first primer with the bisulfite treated gDNA at a first annealing temperature, wherein the first primer comprises:
- (i) a target-complementary portion and (ii) a first primer-binding site upstream from the target-complementary portion;
- (b) extending the annealed first primer to generate a first extension product comprising a nucleotide analog;
- (c) annealing a second primer with the first extension product at a second annealing temperature, wherein the second primer comprises:
- (i) a first extension product-complementary portion and (ii) a second primer-binding site upstream from the first extension product-complementary portion;
- (d) extending the annealed second primer to generate a second extension product comprising a nucleotide analog;
- (e) annealing a third primer with the complement of the first primer-binding site of the second extension product at a third annealing temperature;
- (f) extending the annealed third primer to generate a third extension product;
- (g) annealing a fourth primer with the complement of the second primer-binding site of the third extension product at a fourth annealing temperature;
- (h) extending the annealed fourth primer to generate an additional second extension product;
- and
- (i) optionally, repeating steps (e)-(h) at least one additional cycle.
A method of reducing bias in a PCR amplification, wherein at least two versions of a target genomic locus are present, wherein the two versions differ in their nucleotide composition, the method comprising generating a first extension product, a second extension product, a third extension product, or combinations thereof, in a reaction composition comprising (a) a nucleotide analog and (b) a tailed first primer, a tailed second primer, or a tailed first primer pair.
A kit comprising sodium bisulfite, a polymerase, and a tailed primer pair comprising (1) a first primer comprising (i) a target-complementary portion and (ii) a tail comprising a first primer-binding site upstream from the target-complementary portion and (2), and a second primer comprising (i) a first extension product-complementary portion and (ii) a tail comprising a second primer-binding site upstream from the first extension product-complementary portion.
Dependencies are inferred from the claim text.