Synthesis method of phase codeoxyribonucleic acid

Synthesis method of phase codeoxyribonucleic acid

  • CN 1,246,471 C
  • Filed: 04/10/2001
  • Issued: 03/22/2006
  • Est. Priority Date: 04/10/2001
  • Status: Active Grant
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 " random DNA of representative or RNA sequence comprise 25~

    35 bases, are the combining sites 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 The PolyA tail5’

    ------------- ****xxxxxxxxxxxxxxxxxxxxxxxxxxxxxAAAAAAAAAA3’

    3’



    ----------- ****yyyyyyyyyyyy?yyyyyyyyyyyyyyyyT?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

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