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  • PCR Short

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    Dr. Beenish Aftab
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    The document summarizes the process of reverse transcription polymerase chain reaction (RT-PCR), which is used to convert mRNA to double-stranded cDNA. The process involves using an oligo dT primer to synthesize a first cDNA strand from mRNA, then using that cDNA as a template to synthesize the complementary second strand. The resulting double-stranded cDNA is then amplified through multiple rounds of PCR involving denaturing, annealing of primers, and polymerization to exponentially increase the number of cDNA copies.

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    The document summarizes the process of reverse transcription polymerase chain reaction (RT-PCR), which is used to convert mRNA to double-stranded cDNA. The process involves using an oligo dT primer to synthesize a first cDNA strand from mRNA, then using that cDNA as a template to synthesize the complementary second strand. The resulting double-stranded cDNA is then amplified through multiple rounds of PCR involving denaturing, annealing of primers, and polymerization to exponentially increase the number of cDNA copies.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
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    3 views12 pages

    PCR Short

    Uploaded by

    Dr. Beenish Aftab
    The document summarizes the process of reverse transcription polymerase chain reaction (RT-PCR), which is used to convert mRNA to double-stranded cDNA. The process involves using an oligo dT primer to synthesize a first cDNA strand from mRNA, then using that cDNA as a template to synthesize the complementary second strand. The resulting double-stranded cDNA is then amplified through multiple rounds of PCR involving denaturing, annealing of primers, and polymerization to exponentially increase the number of cDNA copies.

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    © All Rights Reserved
    Download as PPT, PDF, TXT or read online from Scribd
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    of 12

    AAAAA Oligo dT primer is

    RT TTTTT bound to mRNA

    Reverse
    AAAAA transcriptase
    RT (RT) copies first
    TTTTT
    cDNA strand

    Reverse
    AAAAA RT
    transcriptase
    TTTTT
    digests and
    displaces mRNA
    and copies
    second strand of
    cDNA

    Double
    strand
    cDNA

    Conversion of mRNA to cDNA by Reverse Transcription


    A. Double
    50º strand DNA

    96º B. Denature

    50º C. Anneal
    primers

    Taq

    72º D. Polymerase
    binds
    Taq
    Taq Taq
    72º E. Copy
    strands
    Taq Taq

    96º
    2 F.
    Denature

    First round 3
    of cDNA
    synthesis (4
    strands) 4
    1

    G. Anneal
    50º primers

    4
    Taq 1

    72º

    Taq
    2

    H.
    Polymerase
    Taq 3 binds

    Taq
    4
    1
    Taq

    72º

    Taq 2

    I. Copy
    strands

    3
    Second Taq
    round of
    cDNA
    synthesis
    (8 strands)
    Taq
    4
    1

    J.
    Denature at 96º
    Anneal primers
    at 50º

    4
    1

    72º
    K. Bind polymerase
    (not shown) and copy
    strands

    Third
    3
    round of
    cDNA
    synthesis
    (16
    strands)

    4
    1

    L.
    Denature at 96º
    Anneal primers
    at 50º

    2
    3

    4
    1
    M.
    Copy strands at
    72º 72º

    2
    3
    Fourth
    round of
    cDNA
    synthesis
    (32
    strands)

    4
    1

    cDNA
    strands
    (32) are
    now
    shown as
    lines 2
    3

    4
    1

    After 5 rounds
    there are 32
    double strands of
    which 24 (75%)
    are are same size

    2
    3

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