Methods for analysis of molecular events
AssigneeExact Sciences Corporation
InventorsAnthony P. Shuber, Lisa Kann, Duncan Whitney
Life of this patent
Estimating the term…
Abstract
Methods and compositions are provided for detecting the presence of nucleic acid sequence variants in a subpopulation of nucleic acid molecules in a biological sample. These methods are particularly useful for identifying individuals with mutations indicative of cancer.
Claims33
A method for detecting a variant in a primer extension assay, the method comprising the steps of:
- a) contacting a target nucleic acid with a nucleic acid primer complementary to a region of said target nucleic acid;
- and
- b) extending said primer in the presence of:
- i) a first nucleotide that is complementary to a first variant nucleotide suspected to be at a position downstream of said region;
- and,
- ii) a second nucleotide that is complementary to a second variant nucleotide at said position, thereby to reduce misincorporation of said first nucleotide on a template comprising said second variant nucleotide..
A method for minimizing nucleotide misincorporation in a primer extension assay, the method comprising the steps of:
- a) contacting a target nucleic acid with a nucleic acid primer complementary to a region of said target nucleic acid;
- and
- b) extending said primer in the presence of:
- i) a first nucleotide that is complementary to a first variant base suspected to be at a position downstream of said region, and a first polymerase that preferentially incorporates said first nucleotide;
- and,
- ii) a second nucleotide that is complementary to a second variant base at said position and a second polymerase that preferentially incorporates said second nucleotide, thereby to reduce misincorporation of said first nucleotide in a primer extension reaction at said second variant base.
A method for detecting the presence of a variation at a predetermined position in a heterogeneous nucleic acid sample, the method comprising the steps of:
- a) contacting a target nucleic acid with a nucleic acid primer substantially complementary to said target nucleic acid to form a nucleic acid complex upstream from a predetermined position in said target nucleic acid;
- and
- b) contacting said nucleic acid complex with a first nucleotide, a first polymerase, a second nucleotide, and a second polymerase, wherein said first nucleotide is complementary to a first variant base at said predetermined position, and said second nucleotide is complementary to a second variant base at said predetermined position, and wherein said first and second polymerases are different, thereby to reduce misincorporation of said first nucleotide in a primer extension reaction at a said second variant base.
A kit comprising:
- a) a first nucleotide, a second nucleotide;
- and
- b) a first polymerase adapted for said first nucleotide and a second polymerase adapted for said second nucleotide.
Dependencies are inferred from the claim text.