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Method for nucleic acid amplification by transcription using displacement, and reagents and kit therefor

  • US 5,849,547 A
  • Filed: 04/24/1995
  • Issued: 12/15/1998
  • Est. Priority Date: 07/26/1993
  • Status: Expired due to Fees
First Claim
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1. Method of amplifying a sequence of a target nucleic acid, said sequence comprising, from its 5'"'"' end, in the 5'"'"'-3'"'"' direction, an upstream sequence having at least 5 nucleotides and from its 3'"'"' end in the 3'"'"'-5'"'"' direction, a downstream sequence having at least 5 nucleotides,said method comprising the steps of:

  • obtaining a polynucleotide comprising a first segment corresponding to the sequence to be amplified and at least a second segment comprising the sense sequence of a first RNA polymerase promoter or the antisense sequence of a second RNA polymerase promoter or at least a portion of said sense or antisense sequence, wherein such a second segment comprising said sense sequence of the first promoter or portion thereof is situated upstream of the 5'"'"' end of said first segment, and such a second segment comprising said antisense sequence of the second promoter or portion thereof is situated downstream of the 3'"'"' end of said first segment, andbringing said polynucleotide into contact with an excess amount of a set of primers, in the presence of a system having RNA polymerase activity, RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity and strand displacement activity, under conditions allowing the function of the activities, and in the presence of an excess amount of deoxyribonucleoside triphosphates and ribonucleoside triphosphates, such that said sequence of the target nucleic acid is amplified, said set of primers comprising;

    a) at least one primer selected from the group consisting of;

    1) a first primer comprising successively from its 5'"'"' end towards its 3'"'"' end;

    a first optional polynucleotide segment of an arbitrary sequence comprising at least 5 nucleotides,a second segment comprising at least a portion of the sense sequence of said first RNA polymerase promoter including its 3'"'"'-terminal portion,and a third segment having the same length as said upstream sequence and being either homologous to said upstream sequence, or capable of hybridizing with said upstream sequence, and2) a second primer comprising successively, from its 5'"'"' end towards its 3'"'"' end;

    a first optional segment of an arbitrary sequence comprising at least 5 nucleotides,a second segment comprising at least a portion of the sense sequence of said second RNA polymerase promoter including its 3'"'"'-terminal portion,and a third segment having the same length as said downstream sequence and being either homologous to said downstream sequence, or capable of hybridizing with said downstream sequence,wherein, when said first and second primers are both present, the third segment of one of the first and second primers is homologous to one of the upstream or downstream sequences of said sequence to be amplified and the third segment of the other primer is capable of hybridizing with the other downstream or upstream sequence, andb) at least one primer selected from the group consisting of;

    1) a third primer comprising at its 3'"'"'-end;

    either a sequence containing a segment that is homologous to all or part of the second segment of the first primer and containing the 5'"'"'-end of said sense sequence of said first RNA polymerase promoter,or a sequence homologous to a portion of the first segment of the first primer but not comprising its 5'"'"' end,2) a fourth primer comprising at its 3'"'"'-end;

    either a sequence containing a segment that is homologous to all or part of the second segment of the second primer and containing the 5'"'"'-end of said sense sequence of said second RNA polymerase promoter,or a sequence homologous to a portion of the first segment of the second primer but not comprising its 5'"'"'-end,3) a fifth primer comprising at its 3'"'"'-end;

    either a sequence homologous to a portion of the third primer, said portion not comprising the 3'"'"'-terminal nucleotide of said third primer, or a sequence homologous to at least a portion of said first segment of the first primer,wherein, when said third and fifth primers are both present, the 3'"'"'-end of the third primer hybridizes downstream of the 3'"'"'-end of the fifth primer, and4) a sixth primer comprising at its 3'"'"'-end;

    either a sequence homologous to a portion of the fourth primer, said portion not comprising the 3'"'"'-terminal nucleotide of the fourth primer,or a sequence homologous to at least a portion of said first segment of the second primer,wherein, when said fourth and sixth primers are both present, the 3'"'"'-end of the fourth primer hybridizes downstream of the 3'"'"'-end of the sixth primer.

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