# US 6,297,010 B1 · Method for detecting and identifying mutations > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,297,010 B1 - Document ID: US6297010B1 - Status: Granted - Kind: B1 - Application number: 09/016,542 - Filed: 1998-01-30 - Granted: 2001-10-02 - Earliest claimed: 1998-01-30 - Assignees: Genzyme Corporation - Inventors: James E. Stefano - Web page: https://patentgrep.com/patent/US6297010B1 ## CPC - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/686: Polymerase chain reaction [PCR] - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract This invention provides various methods for identifying one or more genetic alterations in a sample polynucleotide strand. ## Claims ### Claim 1 A method for identifying one or more genetic alterations in a sample polynucleotide strand, comprising: (a) providing a duplex comprising the sample polynucleotide strand and a reference polynucleotide strand; (b) contacting the duplex with an agent which recognizes and protects base pair mismatches under conditions which allow the agent to bind to and protect the duplex at the mismatch to form a duplex:agent complex; (c) removing unprotected base pairs; (d) providing a preselected site from which to sequence; and (e) determining the sequence of the sample strand to identify the one or more genetic alterations in the sample polynucleotide strand. ### Claim 2 (depends on 1) The method according to claim 1, wherein step (d) comprises contacting the duplex with an agent having DNA polymerase activity and a mixture of 2 to 3 different deoxynucleoside triphosphates. ### Claim 3 (depends on 1) The method according to claim 1, wherein step (d) comprises ligating an adapter oligonucleotide to the sample stand. ### Claim 4 (depends on 1) The method according to claim 1 which further comprises step (a)(i), which comprises immobilizing the duplex to a solid support. ### Claim 5 A method for identifying one or more genetic alterations in a sample polynucleotide strand, comprising: (a) providing a plurality of duplexes, wherein each duplex comprises a sample polynucleotide strand and a reference polynucleotide strand; (b) contacting the duplexes with an agent which recognizes and protects base pair mismatches under conditions which allow the agent to bind to and protect the duplexes at the mismatch to form duplex:agent complexes; (c) removing unprotected base pairs; (d) providing a preselected site from which to sequence; and (e) determining the sequence of the sample strands to identify the one or more genetic alterations in the sample polynucleotide strands. ### Claim 6 (depends on 5) The method according to claim 5, wherein step (d) comprises contacting the duplexes with an agent having DNA polymerase activity and a mixture of 2 to 3 different deoxynucleoside triphosphates. ### Claim 7 (depends on 5) The method according to claim 5, wherein step (d) comprises ligating an adapter oligonucleotide to the sample stands. ### Claim 8 (depends on 5) The method according to claim 5, which further comprises step (a)(i), which comprises immobilizing the duplex to a solid support. ### Claim 9 A method for identifying one or more genetic alteration(s) in a sample nucleotide strand comprising: (a) providing a duplex by immobilizing the sample polynucleotide strand on one or more solid supports and contacting the sample polynucleotide strand with a reference polynucleotide strand under conditions suitable to form a duplex between the sample and reference strands; (b) contacting the duplex with an agent which recognizes and protects base pair mismatches under conditions which allow the agent to bind to and protect the duplex at the mismatch to form a duplex:agent complex; (c) removing unprotected base pairs; (d) providing a preselected site from which to sequence; and (e) sequencing the sample polynucleotide strands to identify the one or more genetic alteration(s). ### Claim 10 (depends on 9) The method according to claim 9, wherein step (d) comprises contacting the duplex with an agent having DNA polymerase activity and a mixture of 2 to 3 different deoxynucleoside triphosphates. ### Claim 11 (depends on 9) The method according to claim 9, wherein step (d) comprises ligating an adapter oligonucleotide to the sample stand. ### Claim 12 A method for identifying one or more genetic alteration(s) in one or more sample polynucleotide strands comprising: (a) providing a plurality of duplexes by immobilizing a plurality of sample polynucleotide strands on one or more solid supports and contacting the plurality of sample polynucleotide strands with a reference polynucleotide strand under conditions suitable to form a duplex between the sample and reference strands; (b) contacting the duplex with an agent which recognizes and protects base pair mismatches under conditions which allow the agent to bind to and protect the duplex at the mismatch to form a duplex:agent complex; (c) removing unprotected base pairs; (d) providing a preselected site from which to sequence; and sequencing the sample polynucleotide strands to identify the alteration(s). ### Claim 13 (depends on 12) The method according to claim 12, wherein step (d) comprises contacting the duplex with an agent having DNA polymerase activity and a mixture of 2 to 3 different deoxynucleoside triphosphates. ### Claim 14 (depends on 12) The method according to claim 12, wherein step (d) comprises ligating an adapter oligonucleotide to the sample stand. ### Claim 15 (depends on 1, 5, 9, 12) The method according to any of claims 1, 5, 9 or 12, wherein step (d) comprises ligating two adapter oligonucleotides to the product(s) of step (c). ### Claim 16 (depends on 15) The method according to claim 15, wherein the two adapter oligonucleotides are single-stranded. ### Claim 17 (depends on 15) The method according to claim 15, wherein following ligation of the adapter oligonucleotides, any remaining strands are degraded and extended upon the adapter template to produce a double-stranded products. ### Claim 18 (depends on 1, 5, 9, 12) The method according to any of claims 1, 5, 9 or 12, wherein the agent is MutS. ### Claim 19 (depends on 1, 5, 9, 12) The method according to any of claims 1, 5, 9 or 12, wherein the reference strand further comprises a biotin or analog thereof at the 5′ termini. ### Claim 20 (depends on 1, 5, 9, 12) The method according to any of claims 1, 5, 9 or 12, wherein the reference strand is selected from the group consisting of a PCR product, a multiplex restriction product, a cDNA, and a mRNA. ### Claim 21 (depends on 1, 5, 9, 12) The method according to any of claims 1, 5, 9 or 12, wherein the sample strand is selected from the group consisting of a PCR product, a multiplex restriction product, a cDNA, and a mRNA.