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US 2006/0141523 A1Published application
Integrated method for PCR cleanup and oligonucleotide removal
AssigneeApplera Corporation
InventorsLawrence Greenfield, Douglas Bost
Life of this patent
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Abstract
A method is provided for purifying a desired polynucleotide product by removing unincorporated oligonucleotides from a polymerase or ligase reaction mixture.
Claims41
1-62. (canceled)
A method for removing unincorporated oligonucleotides from a reaction mixture, the method comprising:
- (a) forming a mixture comprising:
- (i) a nucleic acid ligase;
- (ii) a nuclease;
- (iii) a downstream oligonucleotide having a 3′ portion and a 5′ portion, wherein the 5′ portion comprises a 5′ recognition group and a 5′ terminal nucleotide;
- and
- (iv) a template nucleic acid;
- wherein (i) and (ii) are the same or separate enzyme complexes;
- (b) digesting the 5′ portion of the downstream oligonucleotide with the nuclease;
- (c) ligating the digested downstream oligonucleotide to an upstream oligonucleotide with the ligase, wherein the ligating forms a polynucleotide product;
- and
- (d) contacting the mixture with a substrate comprising binding groups that bind the 5′ recognition group, to remove unincorporated downstream oligonucleotides from the reaction mixture.
8 more elements
Dependencies are inferred from the claim text.