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AssigneeCornell Research Foundation, Inc.
InventorsFrancis Barany, Weiguo Cao, Jianmin Huang, Jing Lu
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The present invention is a method for detecting DNA sequence differences including single nucleotide mutations or polymorphisms, one or more nucleotide insertions, and one or more nucleotide deletions. Labeled heteroduplex PCR fragments containing base mismatches are prepared. Endonuclease cleaves the heteroduplex PCR fragments both at the position containing the variation (one or more mismatched bases) and to a lesser extent, at non-variant (perfectly matched) positions. Ligation of the cleavage products with a DNA ligase corrects non-variant cleavages and thus substantially reduces background. This is then followed by a detection step in which the reaction products are detected, and the position of the sequence variations are determined.
A method for identifying a mutant nucleic acid sequence differing by one or more single-base changes, insertions, or deletions, from a normal target nucleotide sequences, said method comprising:
A method according to claim 38, wherein the gene is selected from the group consisting of Bcl2, Mdm2, Cdc25A, Cyclin D1, Cyclin E1, Cdk4, survivin, HSP27, HSP70, p53, p21Cip, p16Ink4a, p19ARF, p15INK4b, p27Kip, Bax, growth factors, EGFR, Her2-neu, ErbB-3, ErbB-4, c-Met, c-Sea, Ron, c-Ret, NGFR, TrkB, TrkC, IGF1R, CSF1R, CSF2, c-Kit, AXL, Flt-1 (VEGFR-1), Flk-1 (VEGFR-2), PDGFRα, PDGFRβ, FGFR-1, FGFR-2, FGFR-3, FGFR-4, other protein tyrosine kinase receptors, β-catenin, Wnt(s), Akt, Tcf4, c-Myc, n-Myc, Wisp-1, Wisp-3, K-ras, H-ras, N-ras, c-Jun, c-Fos, PI3K, c-Src, Shc, Raf1, TGFβ, and MEK, E-Cadherin, APC, TβRII, Smad2, Smad4, Smad 7, PTEN, VHL, BRCA1, BRCA2, ATM, hMSH2, hMLH1, hPMS1, hPMS2, and hMSH3.
A method for identifying a mutant nucleic sequence differing by one or more single-base changes, insertions, or deletions from a normal target nucleic acid sequence, said method comprising:
A thermostable endonuclease which preferentially nicks or cleaves heteroduplexed DNA at a location where base pairs are mismatched or one base beyond the mismatch and generates ends which are suitable for ligation when nicking perfectly matched DNA.
A thermostable endonuclease which preferentially nicks or cleaves heteroduplexed DNA at A/A, G/G, T/T, A/G, A/C, G/A, G/T, T/G, T/C, C/A, or C/T mismatched base pairs at a location where the base pairs are mismatched or one base beyond the mismatch and generates ends which are suitable for ligation when nicking perfectly matched DNA.
A thermostable endonuclease which preferentially nicks or cleaves at least one heteroduplex formed for any single base mutation or polymorphism, except those having gRcg, rcRc, cgYc, or gYgy sequences, where the position of the mismatch is underlined and shown in upper case, and generates ends which are suitable for ligation when nicking perfectly matched DNA.
A thermostable endonuclease which preferentially nicks or cleaves heteroduplexed DNA containing one, two, and three base insertions or deletions, at a location where the base pairs are mismatched or one base beyond the unpaired bases, and generates ends which are suitable for ligation when nicking DNA at perfect matched DNA.
A mutant endonuclease which preferentially nicks or cleaves at least one heteroduplexed DNA, containing mismatched bases, better than a wild-type endonuclease.
A mutant endonuclease V from Thermotoga maritima containing either:
A method for identifying a mutant nucleic acid sequence differing by one or more single-base changes, insertions, or deletions, from a normal target nucleic acid sequence, said method comprising:
Dependencies are inferred from the claim text.