Nucleic acid target generation

Claim 1

A method for generating an amplifying a nucleic acid fragment which comprises: (a) specifically hybridizing a first primer 5' to target nucleic acid sequence, the first primer containing a restriction enzyme recognition sequence 5' to a target binding region, (b) simultaneously with (a), hybridizing a second primer 5' to the first primer, (c) extending the first and second primer so that the first primer extension product is displaced from the target nucleic acid sequence by extension of the second primer, (d) making the first primer extension product double stranded by synthesizing a complementary strand, and (e) amplifying the first primer extension product in an amplification reaction in which the restriction enzyme recognition site of the double stranded nucleic acid fragment is nicked by a restriction enzyme.

Claim 2

A method for generating an amplifying a nucleic acid fragment which comprises: (a) specifically hybridizing a first primer 5' to target nucleic acid sequence, the first primer containing a restriction enzyme recognition sequence 5' to a target binding region, (b) simultaneously with (a), hybridizing a second primer 5' to the first primer, (c) extending the first and second primer so that the first primer extension product is displaced from the target nucleic acid sequence by extension of the second primer, (d) making the first primer extension product double stranded by synthesizing a complementary strand, and (e) amplifying the first primer extension product in an amplification reaction in which the restriction enzyme recognition site of the double stranded nucleic acid fragment is nicked by a restriction enzyme.

Claim 7

A method for generating and amplifying a nucleic acid fragment which comprises: (a) specifically hybridizing two single stranded primers (S1 and S2) containing HincII recognition sequences near their 5' ends to opposite nucleic acid strands flanking a double stranded target nucleic acid sequence, (b) simultaneously with (a), hybridizing two single stranded primers (B1 and B2) to opposite strands of the target nucleic acid sequence at a position 5' to the primers in (a), (c) extending all four primers in the presence of dATP (αS) such that extension of B1 and B2 displaces a first extension product of each of S1 and S2, (d) hybridizing S1, S2, B1 and B2 to the first extension products of S1 and S2 and extending the primers in the presence of dATP (αS) such that extension of B1 and B2 displaces a second extension product of each of S1 and S2, (e) making the second extension products double stranded by synthesizing complementary strands, and (f) amplifying the second extension products in an amplification reaction in which the double stranded HincII recognition sequence is nicked by HincII.

Claim 8

A method for generating and amplifying a nucleic acid fragment which comprises: (a) specifically hybridizing two single stranded primers (S1 and S2) containing HincII recognition sequences near their 5' ends to opposite nucleic acid strands flanking a double stranded target nucleic acid sequence, (b) simultaneously with (a), hybridizing two single stranded primers (B1 and B2) to opposite strands of the target nucleic acid sequence at a position 5' to the primers in (a), (c) extending all four primers in the presence of dATP (αS) such that extension of B1 and B2 displaces a first extension product of each of S1 and S2, (d) hybridizing S1, S2, B1 and B2 to the first extension products of S1 and S2 and extending the primers in the presence of dATP (αS) such that extension of B1 and B2 displaces a second extension product of each of S1 and S2, (e) making the second extension products double stranded by synthesizing complementary strands, and (f) amplifying the second extension products in an amplification reaction in which the double stranded HincII recognition sequence is nicked by HincII.