Methods and compositions for isolating nucleic acid sequence variants
First Claim
1. A method of eluting a polynucleotide from a capture primer, comprising:
- using a strand displacing nucleic acid polymerase to initiate nucleic acid synthesis on a polynucleotide hybridized to a capture primer via a capture primer binding site in the polynucleotide, wherein nucleic acid synthesis is initiated upstream of the capture primer binding site on the polynucleotide and proceeds through the capture primer binding site, thereby eluting the polynucleotide from the capture primerwherein the method comprises sorting a mixture of differentially tagged polynucleotides according the identity of their respective nucleic acid tags.
2 Assignments
0 Petitions
Accused Products
Abstract
The invention is drawn to isolating sequence variants of a genetic locus of interest using a modified iterative primer extension method. The nucleic acids analyzed are generally single stranded and have a reference sequence which is used as a basis for performing iterative single nucleotide extension reactions from a hybridized polymerization primer. The iterative polymerization reactions are configured such that polymerization of the strand will continue if the sequence of the nucleic acid being analyzed matches the reference sequence, whereas polymerization will be terminated if the nucleic acid being analyzed does not match the reference sequence. Nucleic acid strands that have mutations can be isolated using a variety of methods and sequenced to determine the precise identity of the mutation/polymorphism. By performing the method on both strands of the nucleic acid being analyzed, virtually all possible mutations can be identified.
130 Citations
15 Claims
-
1. A method of eluting a polynucleotide from a capture primer, comprising:
-
using a strand displacing nucleic acid polymerase to initiate nucleic acid synthesis on a polynucleotide hybridized to a capture primer via a capture primer binding site in the polynucleotide, wherein nucleic acid synthesis is initiated upstream of the capture primer binding site on the polynucleotide and proceeds through the capture primer binding site, thereby eluting the polynucleotide from the capture primer wherein the method comprises sorting a mixture of differentially tagged polynucleotides according the identity of their respective nucleic acid tags. - View Dependent Claims (2, 3, 4, 5, 6)
-
-
7. A method of sorting a mixture of differentially tagged nucleic acid molecules according to the identity of their corresponding nucleotide tags, the method comprising:
-
(i) contacting a nucleic acid sample comprising a mixture of differentially tagged nucleic acid molecules to a capture primer under hybridization conditions, wherein the capture primer is immobilized on a substrate and comprises a sequence complementary to a capture primer binding site in each of the differentially tagged nucleic acid molecules, whereby the differentially tagged nucleic acid molecules are immobilized; (ii) contacting the immobilized differentially tagged nucleic acid molecules to a polymerization primer under hybridization conditions, wherein the polymerization primer comprises a sequence complementary to a first unique region in the tag of one or more first nucleic acid molecules of the differentially tagged nucleic acid molecules, wherein the polymerization primer binds to the same strand of the one or more first nucleic acid molecules as the capture primer at a location that is 5′
to the capture primer; and(iii) isolating the one or more first nucleic acid molecules by contacting the immobilized differentially tagged nucleic acid molecules to a displacing nucleotide polymerase under nucleic acid polymerization conditions, wherein extension of the polymerization primer displaces the one or more first nucleic acid molecules from the immobilized capture primer; wherein the mixture of differentially tagged nucleic acid molecules are sorted according to the identity of their corresponding nucleotide tags. - View Dependent Claims (8, 9, 10, 11, 12, 13)
-
-
14. A method of sorting a mixture of nucleic acid molecules according to the presence of a unique nucleotide sequence, the method comprising:
-
(i) contacting a sample comprising a mixture of nucleic acid molecules to a capture primer under hybridization conditions, wherein the capture primer is immobilized on a substrate and comprises a sequence complementary to a capture primer binding site present in each of the nucleic acid molecules in the mixture, whereby the nucleic acid molecules in the mixture are immobilized; (ii) contacting the immobilized nucleic acid molecules to a polymerization primer under hybridization conditions, wherein the polymerization primer comprises a sequence complementary to a unique sequence present in one or more first nucleic acid molecules in the mixture, wherein the polymerization primer binds to the same strand of the one or more first nucleic acid molecules as the capture primer at a location that is 5′
to the capture primer; and(iii) isolating the one or more first nucleic acid molecules by contacting the immobilized nucleic acid molecules to a strand displacing nucleotide polymerase under nucleic acid polymerization conditions, wherein extension of the polymerization primer displaces the one or more first nucleic acid molecules from the immobilized capture primer; wherein the nucleic acid molecules are sorted according to the presence of the unique sequence. - View Dependent Claims (15)
-
Specification