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    BT322 Lecture 2 - CDNA Synthesis

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    Gana Khaled
    This document summarizes the process of cDNA synthesis from RNA. It involves: 1) Reverse transcription of RNA into complementary DNA using the reverse transcriptase enzyme. 2) Amplification of the resulting cDNA using PCR for use in downstream processes. 3) The reverse transcription reaction requires a template RNA, primers, nucleotides, buffer, magnesium ions, reverse transcriptase, and water. It can be done in one or two separate steps.

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    BT322 Lecture 2 - CDNA Synthesis

    Uploaded by

    Gana Khaled
    12 views14 pages
    This document summarizes the process of cDNA synthesis from RNA. It involves: 1) Reverse transcription of RNA into complementary DNA using the reverse transcriptase enzyme. 2) Amplification of the resulting cDNA using PCR for use in downstream processes. 3) The reverse transcription reaction requires a template RNA, primers, nucleotides, buffer, magnesium ions, reverse transcriptase, and water. It can be done in one or two separate steps.

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    This document summarizes the process of cDNA synthesis from RNA. It involves: 1) Reverse transcription of RNA into complementary DNA using the reverse transcriptase enzyme. 2) Amplification of the resulting cDNA using PCR for use in downstream processes. 3) The reverse transcription reaction requires a template RNA, primers, nucleotides, buffer, magnesium ions, reverse transcriptase, and water. It can be done in one or two separate steps.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    12 views14 pages

    BT322 Lecture 2 - CDNA Synthesis

    Uploaded by

    Gana Khaled
    This document summarizes the process of cDNA synthesis from RNA. It involves: 1) Reverse transcription of RNA into complementary DNA using the reverse transcriptase enzyme. 2) Amplification of the resulting cDNA using PCR for use in downstream processes. 3) The reverse transcription reaction requires a template RNA, primers, nucleotides, buffer, magnesium ions, reverse transcriptase, and water. It can be done in one or two separate steps.

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    © All Rights Reserved
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    cDNA Synthesis

    cDNA Synthesis
     Reverse Transcription of RNA into complementary DNA

     Amplification of cDNA, using PCR, to be used in downstream


    processes

     Can be performed in separate two steps, or combined into one


    Reverse Transcription
    • Reverse Transcriptase enzyme

    • It’s an RNA-dependent DNA polymerase

    • Polymerases are enzymes that are able to string together


    individual DNA building blocks to form long molecular
    strands

    • Used by certain retroviruses


    Reverse Transcription
     Polymerases require:
    1. DNA building blocks; nucleotides
    2. Single-stranded template
    3. A small fragment of DNA, primer, to which they attach
    and start amplification
    4. Temperature
    Reverse Transcription
     Reaction components:

    1. Template is mRNA (typically 1 – 5% of total RNA for a


    mammalian cell)
    Reverse Transcription
    2. Primers:

    a. Oligo(dT)12-18:binds to the endogenous poly (A)+ tail


    at the 3' end of mammalian mRNA’s
    Reverse Transcription
    2. Primers:
    b. Random Hexanucleotides: bind to the mRNA at a
    variety of complementary sites.
     Ideal for overcoming the difficulties presented by extensive
    secondary structures
     Transcribe 5’ regions of mRNA more efficiently
    Reverse Transcription
    2. Primers:

    c. Specific oligonucleotide primers: selectively primes


    the mRNA of interest
    Reverse Transcription
     Design of reverse transcription primers
    1. Primers anneal to sequences on exons on both sides of an
    intron
    Reverse Transcription
     Design of reverse transcription primers
    2. Primers span exon/exon boundaries
    Reverse Transcription
    3. Buffer with Mg2+ ions
    4. dNTPS
    5. Reverse transcriptase
    6. DEPC-treated water
    7. *Optional: RNase inhibitor
    First Strand cDNA Synthesis Protocol
    1

    1 2
    Any questions ?

    Thank you!

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