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    PCR

    Uploaded by

    Nopiyana Pujiastuti
    The document discusses Polymerase Chain Reaction (PCR), including: - PCR was invented in 1983 by Kary Mullis to amplify specific DNA sequences. He received the Nobel Prize in Chemistry for this work. - PCR uses DNA polymerase to exponentially replicate a targeted DNA region through repeated heating and cooling cycles. - The key steps in each PCR cycle are DNA melting, primer annealing, and primer extension. Multiple cycles lead to exponential amplification of the target DNA. - PCR has many applications including disease detection, paternity testing, GMO testing, forensics, and production of recombinant products like vaccines.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd

    PCR

    Uploaded by

    Nopiyana Pujiastuti
    106 views42 pages
    The document discusses Polymerase Chain Reaction (PCR), including: - PCR was invented in 1983 by Kary Mullis to amplify specific DNA sequences. He received the Nobel Prize in Chemistry for this work. - PCR uses DNA polymerase to exponentially replicate a targeted DNA region through repeated heating and cooling cycles. - The key steps in each PCR cycle are DNA melting, primer annealing, and primer extension. Multiple cycles lead to exponential amplification of the target DNA. - PCR has many applications including disease detection, paternity testing, GMO testing, forensics, and production of recombinant products like vaccines.

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    Pcr

    Original Title

    Pcr

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    The document discusses Polymerase Chain Reaction (PCR), including: - PCR was invented in 1983 by Kary Mullis to amplify specific DNA sequences. He received the Nobel Prize in Chemistry for this work. - PCR uses DNA polymerase to exponentially replicate a targeted DNA region through repeated heating and cooling cycles. - The key steps in each PCR cycle are DNA melting, primer annealing, and primer extension. Multiple cycles lead to exponential amplification of the target DNA. - PCR has many applications including disease detection, paternity testing, GMO testing, forensics, and production of recombinant products like vaccines.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    106 views42 pages

    PCR

    Uploaded by

    Nopiyana Pujiastuti
    The document discusses Polymerase Chain Reaction (PCR), including: - PCR was invented in 1983 by Kary Mullis to amplify specific DNA sequences. He received the Nobel Prize in Chemistry for this work. - PCR uses DNA polymerase to exponentially replicate a targeted DNA region through repeated heating and cooling cycles. - The key steps in each PCR cycle are DNA melting, primer annealing, and primer extension. Multiple cycles lead to exponential amplification of the target DNA. - PCR has many applications including disease detection, paternity testing, GMO testing, forensics, and production of recombinant products like vaccines.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 42

    PCR

    Polymerase Chain Reaction

    Metode enzimatis untuk melipatgandakan


    secara eksponensial suatu sekuen nukleotida
    tertentu
    It was invented in 1983
    by Dr. Kary Mullis, for
    which he received the
    Nobel Prize in Chemistry
    in 1993.

    PCR
    Metode yg mengamplifikasi atau
    mengkopi sejumlah kecil rangkaian
    DNA atau satu molekul DNA.
    Memisahkan gen-gen berkopi tunggal
    dri sekelompok sekuen genom.

    Why Polymerase?
    It is called polymerase because the only enzyme
    used in this reaction is DNA polymerase.

    Why Chain?

    It is called chain because the products of the


    first reaction become substrates of the following
    one, and so on.

    The Reaction

    PCR tube

    THERMOCYCLER

    PCR Machine / Thermocycler

    Components of PCR
    Template DNA
    dNTPs (dATP, dTTP, dCTP & dGTP)
    primers
    Taq DNA polymerase
    MgCl2
    PCR buffer

    DNA template
    dNTP(deoxynucleotide
    triphosphate)

    MgCl2

    KCl

    The DNA which will be amplified by the PCR


    provide both the energy and nucleosides for the
    synthesis of DNA.
    It is important to add equal amounts of each
    nucleotide (dATP, dTTP, dCTP, dGTP) to the
    mixture to prevent mismatches of bases.
    The role of Mg++ in PCR forms complexes with
    dNTPs that are the actual substrates for Taq
    Polymerase.
    When Mg++ is too low, primers fail to anneal to
    the target DNA.
    When Mg++ is too high, the base pairing becomes
    too strong and the amplicon fails to denature
    completely when you heat to 94C.
    promotes specific annealing of primers to the PCR
    template. K+ binds to phosphate groups on doublestranded DNA, stabilizing primer annealing.

    Primers
    Short pieces of DNA (20-30 bases) that bind to the DNA
    template allowing Taq DNA polymerase enzyme to initiate
    incorporation of the deoxynucleotides.
    Both specific and universal primers can be used.

    Taq DNA polymerase


    A heat stable enzyme that adds the deoxynucleotides to the
    DNA template.

    Buffer
    keeps the mixture at the proper pH so the PCR reaction will
    take place.

    Three major phases in PCR:


    Denaturing (94C)
    Annealing (55C)
    Extension (72C)
    The total time to perform a standard
    PCR is approximately 4 hours.

    Three major phases in PCR:


    Denaturing (94C)
    Annealing (55C)
    Extension (72C)
    The total time to perform a standard
    PCR is approximately 4 hours.

    PCR cycling program


    Denaturation

    At temperatures above 90C, double-stranded DNA denatures or "melts".


    That means the weak hydrogen bonds that usually hold the two
    complementary strands together at normal temperatures are disrupted
    resulting in two single stranded DNA strands

    DNA melting
    The three parts of the polymerase chain reaction are carried out in
    the same vial, but at different temperatures.
    The first part of the process separates the two DNA chains in the
    double helix.
    This is done simply by heating the vial to 95 oC for 30 seconds.

    double-stranded DNA

    single-stranded DNA

    Annealing
    After separating the DNA strands, the temperature is lowered so the
    primers can attach themselves to the single DNA strands. The
    temperature of this stage depends on the primers and is usually 5C
    below their melting temperature (45-60C).

    Primer annealing
    The primers cannot bind to the DNA strands at such a
    high temperature, so the vial is cooled to 55 oC. At
    this temperature, the primers bind or "anneal" to the
    appropriate location in the DNA strands.
    This takes about 20 seconds.

    single-stranded DNA + primer

    annealed DNA

    The final step of the reaction is to make a complete copy of the


    templates.

    Primers
    Primers are short, artificial DNA strands often not more than 50 and
    usually only 18 to 25 base pairs long that are complementary to the
    beginning or the end of the DNA fragment to be amplified
    Examples of bacteria universal primer sequences are:
    Forward 5' GAT CCT GGC TCA GGA TGA AC 3' (20 mer)
    Reverse 5' GGA CTA CCA GGG TAT CTA ATC 3' (21 mer)
    Estimation of the melting and annealing temperatures of primer:
    If the primer is shorter than 25 nucleotides, the approx. melting
    temperature (Tm)
    Tm=4(G+C) + 2 (A+T)
    GAGGTAACCACACCAGA 4Gs, 5Cs, 7As, 1T
    Tm = {4(4+5)}+{2(7+1)}
    = (49)+(28)
    = 36+16
    = 52
    Tm = 52

    Annealing temperature should be approx. 5 lower than the Tm

    Extension
    Finally, the DNA polymerase has to copy the DNA strands.
    It starts at the annealed primer and works its way along the DNA
    strand. The extension temperature depends on the DNA
    polymerase.
    Taq polymerase extends optimally at a temperature of 72C. The
    time for this step depends both on the DNA polymerase itself and
    on the length of the DNA fragment to be amplified

    Primer extension
    The Taq polymerase begins adding nucleotides to the primer and
    eventually makes a complementary copy of the section of the
    template
    that lies between the primers. This completes one PCR cycle.

    primer-annealed DNA
    with Taq polymerase and
    dATP, dTTP, dGTP, dCTP

    primer-extended DNA

    (1) 90C, open the DNA doublestranded helix.


    (2) 50C, hybridization with
    primer.
    (3)75C, DNA synthesize.

    Repeating for 20~30 times.


    The amount doubles in every cycle.

    PCR Animation
    Please click here.

    Process
    Denature

    Anneal Primer

    Replicate
    DNA
    1st cycle

    2nd cycle

    3rd cycle

    Extraction of DNA

    Add protease
    Add lysis buffer

    Vortex each tube for 15 sec. to ensure proper mixing.

    Incubate each tube for 10 min. at 56C.

    Centrifuge each to remove any


    mixture that may be on the lid.

    Add 210l of ethanol,vortex


    and then centrifuge again.

    Collection DNA

    The plate is then placed in the automated reader,where each


    well is read spectrophotometrically.

    Preparation the master mix


    Master Mix
    10x Buffer - 10 l
    MgCl2 - 6 l
    dNTP mix - 0.8 l of each
    nucleotide
    F5F primer - 2 l
    F5R primer - 2 l
    Taq polymerase - 0.5 l
    Sterile H2O - 73.7 l

    Place 5l of sample and 95l of master mix in vials and place


    these vials in a PCR panel, whichwill then be placed in the
    thermocycler for theDNA amplification cycles.

    Designing PCR programs

    APLIKASI PCR
    - Deteksi dini penyakit infeksi dan herediter
    - Test Paternity & Maternity
    - Uji GMO (genetically modified organism)
    - Uji keseragaman/kemurnian genetis
    - Test forensik
    - Peningkatan kualitas & kuantitas produk biologis
    - Pembuatan produk rekombinan (vaksin, hormon,
    dll.)

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