Patent ID: 6063604
Filing Date: 2000-05-16
Classification: C12Q

Abstract:
A method for segregating a copy of a target nucleic acid sequence found within a single-stranded polynucleotide, the segregated copy suitable for use in amplifying the target nucleic acid sequence, the method comprising:(a) providing a reaction mixture comprising:(i) a sample containing or suspected of containing a target nucleic acid sequence positioned within a single-stranded polynucleotide at other than an end of said polynucleotide;(ii) a first polymerizing enzyme which polymerizes deoxyribonucleoside triphosphates by extending a hybridized primer, using the single strand of said polynucleotide as a template, and a second polymerizing enzyme which polymerizes deoxyribonucleoside triphosphates by extending a hybridized primer and further which displaces a strand, with the proviso that when the target nucleic acid is a DNA, the first polymerizing enzyme is optionally the same as the second polymerizing enzyme;(iii) a mixture of deoxyribonucleoside triphosphates that includes a modified deoxyribonucleoside triphosphate; and(iv) a first amplification primer which hybridizes to the target nucleic acid sequence and forms a hybridized primer therewith, the first amplification primer comprising an oligodeoxyribonucleotide having a 3' end and a 5' end, the 3' end of said first amplification primer being complementary to the 3' end of said target nucleic acid sequence, the 5' end of said first amplification primer having a first recognition sequence for a first restriction endonuclease that is capable of nicking one strand of a double-stranded hemi-modified recognition site for said first restriction endonuclease; and(v) a second amplification primer comprising an oligodeoxyribonucleotide having a 3' end and a 5' end, the 3' end of said second amplification primer being complementary to the 3' end of the complement of said target nucleic acid sequence and which hybridizes thereto, the 5' end of said second amplification primer having a second recognition sequence for a second restriction endonuclease that is capable of nicking one strand of a double-stranded hemi-modified recognition site for said second restriction endonuclease;(b) subjecting the reaction mixture to a means for separating any double-stranded nucleic acid in the reaction mixture into single strands which means is a RNase enzyme; and(c) allowing the reaction mixture sufficient time for hybridization, primer extension, and nicking to occur, whereby there is produced a double-stranded polynucleotide extension product suitable for amplification comprising a segregated copy of said target nucleic acid sequence and a segregated copy of the complement of said target nucleic acid sequence, said double-stranded polynucleotide extension product being defined at opposing ends by a first nickable restriction site and a second nickable restriction site for said first and second restriction endonucleases, respectively.