A kind of primer middle part sequence interferes round pcr

A kind of primer middle part sequence interferes round pcr

  • CN 103,114,131 B
  • Filed: 12/17/2012
  • Issued: 10/02/2018
  • Est. Priority Date: 11/30/2012
  • Status: Active Grant
First Claim
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1. sequence synthesis interferes PCR method in the middle part of a kind of primer, it is characterised in that:

  • Target base is carried out under the conditions of PCR primer sustained releaseBecause of PCR amplification, PCR system has following(

    a)





    b)

    With(

    c)

    Feature can not influence target gene and primer full length sequenceIn conjunction with and specific amplification efficiency in the case of selectively inhibit that end combines between primer and primer dimer PD is non-specificAmplification:



    a)

    Including upstream and downstream primer, the middle part series of the upstream and downstream primer are not complementary;

    The middle part seriesNot complementary to refer to the parallel comparison in the middle part directions sequence 5'"'"'-3'"'"', inclined 3'"'"' end positions are that 4-5 from the ends 3'"'"' base plays 5-9 alkali reciprocalBase base is not continuously complementary, and 2 or more reverse complemental bases, 1~2 base of primer 3'"'"' least significant ends is avoided to keep away between the ends primer pair 3'"'"'Any single reverse complemental base between exempting from, end are ended up with base C or A;



    b)

    The upstream and downstream primer further includes the antisense nucleobase oligonucleotide with sequence complementation in the middle part of a primer;

    InstituteAntisense nucleobase oligonucleotide segment is stated, the chemical modification antisense modified base with 5-11 endcapped can neither be useed asPcr template can not be used as primer, remain binding function, can competitively combine and gather between sequence interference primer in the middle part of primerClose, only in the middle part of primer the antisense nucleobase oligonucleotide interference of sequence do not influence primer and combined with target gene specific and spySpecific amplification efficiency, and selectively inhibit optimizational primer PCR primer dimer PD non-specific amplifications;



    c)

    The upstream and downstream primer is connected to using the antisense base sequence with sequence complementation in the middle part of primer before the ends primer 5'"'"'Form the chimeric primers of the ends 3'"'"' binding sequence containing specific target and the ends the 5'"'"' base sequence containing antisense in primer molecule;

    Described and primerThe antisense base sequence of middle part sequence complementation refers to the antisense of sequence area 5-7 base sequences in the middle part of sense primer and/or downstream primerChain base is added in the ends 5'"'"' of the corresponding upstream or downstream primer by the directions 5'"'"'-3'"'"' in a manner of chemical synthesis and is fitted into be formedPrimer, the ends 5'"'"' antisense base sequence energy reflexed are simultaneously combined inhibition with sequence in the middle part of primer itself, can substantially reduce system inner primerDimer PD non-specific amplifications react,The method is non-diagnostic purpose.

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