Synthesis method of phase codeoxyribonucleic acid

Synthesis method of phase codeoxyribonucleic acid

  • CN 1,380,421 A
  • Filed: 04/10/2001
  • Published: 11/20/2002
  • Est. Priority Date: 04/10/2001
  • Status: Abandoned Application
First Claim
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1. , the synthetic method of a kind of cDNA is characterized in that:

  • comprise the following steps;

    (1) DNA-RNA or the RNA donor molecule of usefulness ordinary method synthetic following structural;

    5 '"'"'------------------- * * *3 '"'"', comprise ①

    5’

    OOOOOOOOOOOOOO-GNGG3’



    5’

    OOOOOOOOOOOOOO-NGGG3’



    5’

    OOOOOOOOOOOOOO-NNGG3’



    5’

    OOOOOOOOOOOOOO-GNG3’



    5’

    OOOOOOOOOOOOOO-GGN3’



    5’

    OOOOOOOOOOOOOO-NGG3’

    Wherein Poly "

    O "

    represents random DNA or RNA sequence, comprises 25~

    35 bases, is the combining site of primer;

    " G " represents the RNA bases G, and " N " represents RNA base;

    A or T, G, C;

    (2) adopting ordinary method with murine reverse transcriptase M-MLV is the synthetic first chain cDNA of template with mRNA, in the synthetic first chain cDNA, add above-mentioned donor molecule, the reverse transcription and the template conversion reaction of following mode take place;

    the 5 '"'"' end that arrives mRNA as the synthetic first chain cDNA, the first chain cDNA holds the cDNA acceptor molecule that has the tailing function and form 3 '"'"', by the auxilliary mutually combination of molecule, donor molecule in this receptor molecule acceptable response liquid, reversed transcriptive enzyme continues donor molecule is continued to transcribe as template and a synthetic sequence auxilliary mutually with donor molecule, i.e. the PCR universal sequence;

    5 '"'"'--------- * * *3 '"'"' donor molecule PolyA tail 5 '"'"'--------------- * * *XxxxxxxxxxxxxxxxxxxxxxxxxxxAAAAAAAA3 '"'"' 3 '"'"' ←

    -------------- * * *Yyyyyyyyy yyyyyyyyyyyyyyyyyT TTT T TTT5 '"'"' Acceptor molecule oligo-d (T) wherein "

    x "

    represents mRNA, and "

    y "

    represents the first chain cDNA;

    (3), and generate a large amount of amplified productions with synthetic cDNA second chain of conventional pcr amplification method.5 '"'"' end primer----→

    5 '"'"'------------------- * * *XxxxxxxxxxxxxxxxxxxxxxxxxxxxxxAAAAAAAAAAA3 '"'"' 3 '"'"'------------------- * * *Yyyyyyyyyyyyyyy yyyyyyyyyyyyyyTT T TTT T T5 '"'"' ←

    -------3 '"'"' end primer

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