Enrichment Through Heteroduplexed Molecules
First Claim
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1. A method for the enrichment of a nucleic acid sequence, said method comprising:
- forming a heteroduplex comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein said heteroduplex comprises at least one mismatched base pair between the first nucleic acid sequence and the second nucleic acid sequence;
circularizing the heteroduplex to form a circularized heteroduplex (CHET);
preferentially cutting the CHET to form a duplex using an enzyme that recognizes the mismatched base pair;
ligating a binding moiety nucleic acid sequence (BMNAS) into the duplex to form a binding moiety duplex, wherein said BMNAS comprises;
two nucleic acid sequences hybridized to one another and a binding moiety; and
selecting the binding moiety by using a purifying moiety to bind to the binding moiety, thereby enriching the second nucleic acid sequence.
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Abstract
The present invention relates to the enrichment of specific target sequences Enrichment can be achieved through the formation of a heteroduplex that includes the specific target sequence and then the specific cleavage of the heteroduplex. A binding moiety is then added to the cleaved heteroduplex, allowing for the subsequent manipulation of the specific target sequence in the heteroduplex.
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Citations
37 Claims
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1. A method for the enrichment of a nucleic acid sequence, said method comprising:
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forming a heteroduplex comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein said heteroduplex comprises at least one mismatched base pair between the first nucleic acid sequence and the second nucleic acid sequence; circularizing the heteroduplex to form a circularized heteroduplex (CHET); preferentially cutting the CHET to form a duplex using an enzyme that recognizes the mismatched base pair; ligating a binding moiety nucleic acid sequence (BMNAS) into the duplex to form a binding moiety duplex, wherein said BMNAS comprises;
two nucleic acid sequences hybridized to one another and a binding moiety; andselecting the binding moiety by using a purifying moiety to bind to the binding moiety, thereby enriching the second nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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37. A method for the enrichment of a nucleic acid sequence, said method comprising:
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providing a sample containing a first nucleic acid sequence (FNAS) as well as a second nucleic acid sequence (SNAS); providing two primers suitable for hybridization on complementary strands of the FNAS and the SNAS; providing a polymerase; combining the sample, the primers, and the polymerase to form a polymerase chain reaction mixture; subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles to create a polymerase chain reaction extension product; denaturing the polymerase chain reaction extension product to separate a FNAS and a SNAS; annealing the polymerase chain reaction extension product to form a heteroduplex comprising the FNAS and the SNAS and to form a homoduplex comprising the FNAS; circularizing the heteroduplex to form a circularized heteroduplex (CHET); circularizing the homoduplex to form a circularized homoduplex (CHOM); providing an endonuclease that preferentially cuts the heteroduplex at a location one base away from mismatched base pairs, wherein said cutting of the heteroduplex is preferential over the cutting of the homoduplex; combining the CHET, the CHOM, and the endonuclease to form an endonuclease cleavage reaction mixture; incubating the endonuclease cleavage reaction mixture so that the endonuclease preferentially cuts the CHET at a location one base away from a mismatched base pairs; providing a plurality of binding moiety nucleic acid sequences (BMNAS), wherein said BMNASs comprise; a double stranded nucleic acid sequence; biotin associated with the double stranded nucleic acid sequence, two EcoP15I restriction sites on each nucleic acid sequence, wherein one restriction site is located near or at the 3′
end of one nucleic acid strand and the second restriction site is located near or at the 5′
end of the same nucleic acid strand, anddegenerate ends on each end of each nucleic acid sequence; ligating the BMNAS into the duplex to form a binding moiety duplex; cutting the binding moiety duplex using EcoP15I; concentrating the binding moiety duplex by using magnetic beads; cutting the concentrated binding moiety duplex by using EcoP15I to form a cut binding moiety duplex (CBMD); adding a first adapter to a first end of the CBMD; adding a second adapter to a second end of the CBMD, wherein the first and second adapters are different; performing emulsion PCR to amplify the CBMD using the first and second adapters as priming sites; and performing highly parallel non-electrophoretic sequencing technique on the CBMD to identify the mismatched base pair.
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Specification