Patent ID: 7709201
Filing Date: 2010-05-04
Classification: B82Y,C12Q

Abstract:
1. A method for identifying one or more mutant nucleotide target sequences differing by one or more single-base changes, insertions, or deletions, from one or more normal nucleotide target sequences, said method comprising: providing one or more sample(s) potentially containing the normal nucleotide target sequences, one or more mutant nucleotide target sequences, or both; providing a group of one or more primary oligonucleotide primer sets, each set comprising (a) a first primary oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion and (b) a second primary oligonucleotide primer, having a target-specific portion and a 5′ upstream secondary primer-specific portion, wherein the first primary oligonucleotide primers of each set in a group contain the same 5′ upstream secondary primer-specific portion and the second oligonucleotide primers of each set in a group contain the same 5′ upstream secondary primer-specific portion; blending the sample, the one or more primary oligonucleotide primer sets, and a polymerase to form one or more primary polymerase chain reaction mixture(s); subjecting the one or more primary polymerase chain reaction mixture(s) to one or more polymerase chain reaction cycles to form primary extension products complementary to the normal nucleotide and mutant nucleotide target sequences present in the sample; providing a group of one or more secondary oligonucleotide primer sets, each set comprising (a) a first secondary oligonucleotide primer which comprises the same sequence as the 5′ upstream secondary primer-specific portion of the first primary oligonucleotide primer and (b) a second secondary oligonucleotide primer which comprises the same sequence as the 5′ upstream secondary primer-specific portion of the second primary oligonucleotide primer; blending the one or more primary polymerase chain reaction mixture(s), the one or more secondary oligonucleotide primer sets, and a polymerase to form one or more secondary polymerase chain reaction mixture(s); subjecting the one or more secondary polymerase chain reaction mixture(s) to one or more polymerase chain reaction cycles to form secondary extension products complementary to the primary extension products; inactivating the polymerase; subjecting the one or more secondary polymerase chain reaction mixture(s) to a process which converts the secondary extension products to a single-stranded form and anneals the single-stranded secondary extension products to form heteroduplexed products potentially comprising nucleic acid molecules which include nucleotide sequences from the normal nucleotide target sequence and from the mutant nucleotide target sequence; providing an endonuclease, which preferentially nicks or cleaves heteroduplexed DNA at a location within one base away from mismatched base pairs; blending the heteroduplexed products and the endonuclease to form an endonuclease cleavage reaction mixture; subjecting the endonuclease cleavage reaction mixture to an endonuclease cleavage reaction so that the endonuclease preferentially nicks or cleaves heteroduplexed products at a location within one base away from mismatched base pairs; providing a ligase; blending the endonuclease cleavage reaction mixture and the ligase to form a ligase resealing reaction mixture; subjecting the ligase resealing reaction mixture to a ligase resealing reaction to seal the nicked heteroduplexed products at perfectly matched base pairs but with substantially no resealing of nicked heteroduplexed products at locations adjacent to mismatched base pairs; separating products after said subjecting the ligase resealing reaction mixture to a ligase resealing reaction by size or electrophoretic mobility or hybridization to capture probes attached to a solid support; and detecting the presence of the normal nucleotide target sequence and the one or more mutant nucleotide target sequence in the sample by distinguishing the separated products.