Patent ID: 8435741
Filing Date: 2013-05-07
Classification: C12Q

Abstract:
1. A nucleic acid amplification method which comprises performing isothermal incubation of a reaction solution wherein said reaction solution comprising said reaction solution is free of any primers having a structure which forms a loop structure, so as to perform a polymerase reaction that initiates from the 3′ end of the primers and thus amplifying the nucleic acid fragment wherein, (i) (a) one of said at least two types of oligonucleotide primers is annealed to a template nucleic acid strand of the nucleic acid fragment, and a synthesis reaction is initiated from the 3′ end of said annealed oligonucleotide primer due to the action of the DNA polymerase so as to synthesize a primer elongation product; (b) one or more oligonucleotide primers other than the oligonucleotide primer of step (a) are caused to enter the double-stranded nucleic acid obtained in step (a) without performing any denaturation of the double-stranded nucleic acid via application of a temperature higher than that in step (a), and then, a synthesis reaction is initiated from the 3′ ends of said one or more oligonucleotide primers by the action of the DNA polymerase so as to synthesize further primer elongation products; (c) the primer of step (a) is annealed again to the nucleic acid liberated in step (b) and a synthesis reaction is then initiated from the 3′ end of said primer by the action of the DNA polymerase, so as to synthesize further primer elongation products; and (d) the double-stranded nucleic acid obtained in step (b) is used again as a template in step (b); or (ii) (a) one of said at least two types of primers is annealed to a template nucleic acid strand of the nucleic acid fragment, and a synthesis reaction is initiated from the 3′ end of said annealed oligonucleotide primer due to the action of the DNA polymerase so as to synthesize a primer elongation product; (b) the oligonucleotide primer of step (a) is caused to enter the double-stranded nucleic acid obtained in step (a) without performing any denaturation of the double-stranded nucleic acid via application of a temperature higher than that in step (a), and then, a synthesis reaction is initiated from the 3′ end of said oligonucleotide primer by the action of the DNA polymerase so as to synthesize a primer elongation product; (c) one or more oligonucleotide primers other than the primer of step (a) are annealed to the nucleic acid liberated in step (b) and a synthesis reaction is then initiated from the 3′ ends of said one or more oligonucleotide primers by the action of the DNA polymerase, so as to synthesize further primer elongation products; and (d) the double-stranded nucleic acid obtained in step (b) is used again as a template in step (b).