Methods for the selection and cloning of nucleic acid molecules free of unwanted nucleotide sequence alterations
First Claim
1. A method for eliminating an amplified nucleic acid molecule having a nucleotide sequence that is mutated, compared with the target nucleotide sequence used to produce the amplified nucleic acid molecule, comprising, (a) obtaining a mixture of duplexes of amplified nucleic acid molecules, wherein the duplex mixture comprises homoduplexes and heteroduplexes, (b) treating the duplex mixture with a single-strand specific nuclease that cleaves a mismatched site within a heteroduplex, and (c) subcloning the nuclease-treated duplex mixture, wherein a cleaved duplex is unsuitable for subcloning.
1 Assignment
0 Petitions
Accused Products
Abstract
The accurate synthesis of nucleic acid molecules is important for use of amplified nucleic acid molecules as hybridization probes, in the regulation of gene expression, as templates for the production of recombinant proteins, as diagnostic probes, and in forensic analyses. Methods are provided to separate nucleic acid molecules that are free of mutations from a population of nucleic acid molecules that contain unwanted nucleotide alternations.
-
Citations
25 Claims
-
1. A method for eliminating an amplified nucleic acid molecule having a nucleotide sequence that is mutated, compared with the target nucleotide sequence used to produce the amplified nucleic acid molecule, comprising,
(a) obtaining a mixture of duplexes of amplified nucleic acid molecules, wherein the duplex mixture comprises homoduplexes and heteroduplexes, (b) treating the duplex mixture with a single-strand specific nuclease that cleaves a mismatched site within a heteroduplex, and (c) subcloning the nuclease-treated duplex mixture, wherein a cleaved duplex is unsuitable for subcloning.
-
5. A method for eliminating an amplified nucleic acid molecule having a nucleotide sequence that is mutated, compared with the target nucleotide sequence used to produce the amplified nucleic acid molecule, comprising,
(a) obtaining a mixture of duplexes of the amplified DNA molecules, wherein the duplex mixture comprises homoduplexes and heteroduplexes, (b) cleaving the duplex mixture with a single-strand specific nuclease that cleaves a mismatched site within a heteroduplex, wherein the cleavage produces an exposed 3′ - OH moiety,
(c) treating the cleaved duplex mixture with a DNA polymerase and nucleotide analog conjugated with an affinity label to synthesize DNA that comprises the affinity label, and (d) incubating the treated duplex mixture of (c) with an affinity label capture molecule that binds the affinity label, thereby eliminating duplexes that comprise the affinity-labeled DNA. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12)
- OH moiety,
-
13. A method for eliminating an amplified nucleic acid molecule having a nucleotide sequence that is mutated, compared with the target nucleotide sequence used to produce the amplified nucleic acid molecule, comprising,
(a) obtaining a mixture of duplexes of the amplified DNA molecules, wherein the duplex mixture comprises homoduplexes and heteroduplexes, (b) treating the duplex mixture with potassium permanganate, tetraethylammonium chloride, and hydroxylamide to create carbonyl groups in mismatched nucleotides, (c) labeling the carbonyl groups of the duplex mixture with hydrazine derivatized with an affinity label, and (d) incubating the labeled duplex mixture of (c) with an affinity label capture molecule that binds the affinity label, thereby eliminating duplexes that comprise the affinity-labeled DNA.
-
19. A method for eliminating an amplified nucleic acid molecule having a nucleotide sequence that is mutated, compared with the target nucleotide sequence used to produce the amplified nucleic acid molecule, comprising,
(a) obtaining a mixture of duplexes of the amplified DNA molecules, wherein the duplex mixture comprises homoduplexes and heteroduplexes, (b) treating the duplex mixture with 1-cyclohexyl-3-{2-[4-(4-methyl)morpholinyl]ethyl }carbodiimide derivatized with an affinity label, and (c) incubating the treated duplex mixture of (c) with an affinity label capture molecule that binds the affinity label, thereby eliminating duplexes that comprise the affinity-labeled DNA.
Specification