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    Polymerase Chain Reaction

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    ansah
    The document summarizes polymerase chain reaction (PCR), beginning with its discovery and development. PCR amplifies a specific region of DNA using DNA polymerase, primers, and repeated heating/cooling cycles. Key steps include DNA denaturation, primer annealing, and extension. PCR has many applications like gene expression analysis, cloning, diagnostics, and forensics due to being fast, automated, sensitive, and producing a high DNA output.

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    Polymerase Chain Reaction

    Uploaded by

    ansah
    119 views24 pages
    The document summarizes polymerase chain reaction (PCR), beginning with its discovery and development. PCR amplifies a specific region of DNA using DNA polymerase, primers, and repeated heating/cooling cycles. Key steps include DNA denaturation, primer annealing, and extension. PCR has many applications like gene expression analysis, cloning, diagnostics, and forensics due to being fast, automated, sensitive, and producing a high DNA output.

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    PCR-Presentation

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    The document summarizes polymerase chain reaction (PCR), beginning with its discovery and development. PCR amplifies a specific region of DNA using DNA polymerase, primers, and repeated heating/cooling cycles. Key steps include DNA denaturation, primer annealing, and extension. PCR has many applications like gene expression analysis, cloning, diagnostics, and forensics due to being fast, automated, sensitive, and producing a high DNA output.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    119 views24 pages

    Polymerase Chain Reaction

    Uploaded by

    ansah
    The document summarizes polymerase chain reaction (PCR), beginning with its discovery and development. PCR amplifies a specific region of DNA using DNA polymerase, primers, and repeated heating/cooling cycles. Key steps include DNA denaturation, primer annealing, and extension. PCR has many applications like gene expression analysis, cloning, diagnostics, and forensics due to being fast, automated, sensitive, and producing a high DNA output.

    Copyright:

    © All Rights Reserved
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    Download as pdf or txt
    of 24

    Polymerase Chain Reaction

    Introduction
    • PCR, polymerase chain reaction, is an in-
    vitro technique for amplification of a region
    of DNA whose sequence is known or
    which lies between two regions of known
    sequence
    • Before PCR, DNA of interest could only be
    amplified by over-expression in cells and
    this with limited yield
    • 1966, Thomas Brock discovers Thermus
    Aquaticus, a thermostable bacteria in the hot
    springs of Yellowstone National Park

    • 1983, Kary Mullis postulated the concept of PCR

    ( Nobel Prize in 1993)

    • 1985, Saiki publishes the first application of PCR (


    beta-Globin)

    • 1985, Cetus Corp. Scientists isolate Thermostable


    Taq Polymerase (from T.Aquaticus), which
    revolutionized PCR
    Reaction Components
    • DNA template
    • Primers
    • Enzyme
    • dNTPs
    • Mg2+
    • buffers
    1- DNA template

    • DNA containing
    region to be
    sequenced
    2- Primers
    • 2 sets of primers
    • Generally 20-30
    nucleotides long
    • Synthetically
    produced
    • complimentary to
    the 3’ ends of target
    DNA
    • not complimentary
    to each other
    Primers
    • Not containing inverted repeat sequences
    to avoid formation of internal structures
    • 40-60% GC content preferred for better
    annealing
    • Tm of primers can be calculated to
    determine annealing T0
    • Tm= .41(%G+C) + 16.6log(J+) + 81.5
    where J+ is the concentration of
    monovalent ions
    3-Enzyme
    • Usually Taq Polymerase or anyone of the
    natural or Recombinant thermostable
    polymerases
    • Stable at T0 up to 950 C
    • High processivity
    The PCR Cycle
    • Comprised of 3 steps:
    - Denaturation of DNA at 950C
    - Primer hybridization ( annealing) at 40-
    500C
    - DNA synthesis ( Primer extension) at
    720C
    Standard thermocycle
    RT-PCR
    Reverse transcriptase-PCR
    • DNA polymerase that is important in genetic
    engineering is reverse transcriptase, an
    enzyme involved in the replication of several
    kinds of virus.
    • Reverse transcriptase is unique in that it uses
    as a template not DNA but RNA.
    • The ability of this enzyme to synthesize a
    DNA strand complementary to an RNA
    template is central to the technique called
    complementary DNA (cDNA) cloning.
    • The quality and purity of the RNA template is essential for
    the success of RT-PCR.
    • The first step of RT-PCR is the synthesis of a DNA/RNA
    hybrid.
    • Reverse transcriptase also has an RNase H function, which
    degrades the RNA portion of the hybrid.
    • The single stranded DNA molecule is then completed by the
    DNA-dependent DNA polymerase activity of the reverse
    transcriptase into cDNA.
    • The efficiency of the first-strand reaction can affect the
    amplification process.
    • From here on, the standard PCR procedure is used to
    amplify the cDNA.
    The possibility to revert RNA into cDNA by
    RT-PCR has many advantages.
    • RNA is single-stranded and very unstable,
    which makes it difficult to work with.
    • Most commonly, it serves as a first step in
    qPCR, which quantifies RNA transcripts in
    a biological sample.
    Detection of amplification
    products
    • Gel electrophoresis
    • Sequencing of amplified fragment
    • Southern blot
    Applications
    • Detection of expressed genes,
    • Examination of transcript variants,
    • Generation of cDNA templates for cloning and
    sequencing.
    • Genome mapping and gene function
    determination
    • Biodiversity studies ( e.g. evolution studies)
    • Diagnostics ( prenatal testing of genetic diseases,
    early detection of cancer, viral infections...)
    • Detection of drug resistance genes
    • Forensic (DNA fingerprinting)
    Advantages
    • Automated, fast, reliable (reproducible)
    results
    • Contained :(less chances of contamination)
    • High output
    • Sensitive
    • Broad uses
    • Defined, easy to follow protocols

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