Scribd Logo
Upload

Welcome to Scribd!

  • PCR Techniques Explained

    Uploaded by

    himawarum
    13 views17 pages
    The document discusses the principles and techniques of polymerase chain reaction (PCR). It begins by explaining the basic components of PCR including DNA template, primers, DNA polymerase and dNTPs. It then describes the three main steps of PCR: denaturation, annealing and extension. The document also discusses primer design considerations and techniques like degenerate primers and inosine bases. It explains variants of PCR including reverse transcriptase PCR (RT-PCR) for amplifying mRNA, nested PCR for increased sensitivity, and RAPD-PCR which uses single random primers.

    Original Description:

    Original Title

    TM 08 POLYMERASE CHAIN REACTION 2014.pptx

    Copyright

    © © All Rights Reserved

    Available Formats

    PPTX, PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    Report this Document
    The document discusses the principles and techniques of polymerase chain reaction (PCR). It begins by explaining the basic components of PCR including DNA template, primers, DNA polymerase and dNTPs. It then describes the three main steps of PCR: denaturation, annealing and extension. The document also discusses primer design considerations and techniques like degenerate primers and inosine bases. It explains variants of PCR including reverse transcriptase PCR (RT-PCR) for amplifying mRNA, nested PCR for increased sensitivity, and RAPD-PCR which uses single random primers.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    13 views17 pages

    PCR Techniques Explained

    Uploaded by

    himawarum
    The document discusses the principles and techniques of polymerase chain reaction (PCR). It begins by explaining the basic components of PCR including DNA template, primers, DNA polymerase and dNTPs. It then describes the three main steps of PCR: denaturation, annealing and extension. The document also discusses primer design considerations and techniques like degenerate primers and inosine bases. It explains variants of PCR including reverse transcriptase PCR (RT-PCR) for amplifying mRNA, nested PCR for increased sensitivity, and RAPD-PCR which uses single random primers.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    of 17

    TATAP MUKA 8

    Polymerase Chain Reaction


    (PCR)

    LO 34: menjelaskan prinsip dasar PCR


    LO 35: menjelaskan perancangan prmer PCR
    LO 36: menjelaskan reverse transcriptase PCR (RT-PCR)
    LO 37: menjelaskan nested PCR
    LO 38: menjelaskan RAPD-PCR

    RINSIP DASAR PCR

    OMPONEN PCR

    1. DNA templat
    DNA yang sebagian urutannya
    akan diamplifikasi
    2. Primer
    Oligonucleotide
    untuk memulai amplifikasi
    membatasi fragmen yang
    akan diamplifikasi.
    3. DNA polimerase
    DNA polimerase termostabil
    mengamplifikasi
    fragmen
    DNA
    4. dNTP
    Monomer DNA
    LO 34: menjelaskan prinsip dasar PCR
    dATP, dTTP, dGTP, dCTP

    LUS DAN TAHAPAN PCR


    DENATURATION (95C)
    Duplex DNA is heat
    denatured to give
    single strands
    ANNEALING (55C)
    two oligonucleotide
    primers are annealed
    to their
    complementary
    sequences on the
    target DNA.
    EXTENSION (72C)
    Taq polymerase
    (thermostable) is used
    to synthesise
    complementary

    LO 34: menjelaskan prinsip dasar PCR

    LUS DAN TAHAPAN PCR

    LO 34: menjelaskan prinsip dasar PCR

    ERANCANGAN PRIMER PCR


    Aspek yang harus dipertimbangkan:
    urutan primer
    asal informasi urutan
    data sekuens asam amino
    kode degenerasi genetik
    Urutan basa DNA yang telah
    diketahui
    Sintesis primer:
    Posisi Wobble
    Basa inosin
    LO 35: menjelaskan perancangan prmer PCR

    ERANCANGAN PRIMER PCR

    Fig. 7.3 Primer design. In (a) the amino acid sequence


    and number of codons per amino acid are shown. Those
    amino acids with 6 codons (circled) are best avoided. The
    boxed sequence is therefore selected.

    LO 35: menjelaskan perancangan prmer PCR

    Fig. 7.3 Primer design. In (b) a mixed probe is synthesised


    by including the appropriate mixture of dNTPs for each
    degenerate position. Note that in this example the final
    degenerate position for proline is not included, giving an
    oligonucleotide with 20 bases (a 20-mer). There are 128
    possible permutations of sequence in the mixture.

    LO 35: menjelaskan perancangan prmer PCR

    Fig. 7.3 Primer design. In (c) inosine (I) is used to replace


    the four-fold degenerate bases, giving eight possible
    sequences.
    LO 35: menjelaskan perancangan prmer PCR

    Varian PCR

    everse transcriptase PCR (RT-PCR)


    PCR menggunakan mRNA sebagai templat
    Digunakan dalam penentuan tingkat ekspresi
    gen
    Proses penyalinan mRNA memerlukan reverse
    transcriptase
    Primer Oligo(dT) digunakan untuk mensintesis
    untai pertama cDNA.
    Primer PCR digunakan setelah untai pertama
    cDNA disintesis

    LO 36: menjelaskan reverse transcriptase PCR (RT-PCR)

    Fig. 7.4 RT-PCR. (a) Reverse transcriptase is used to synthesise a cDNA


    copy of the mRNA. In this example oligo(dT)-primed synthesis is shown.
    (b) The cDNA product is amplified using genespecific primers. The initial
    PCR synthesis will copy the cDNA to give a duplex molecule, which is then
    amplified in the usual way. In many kits available for RT-PCR the entire
    procedure can be carried out in a single tube.
    LO 36: menjelaskan reverse transcriptase PCR (RT-PCR)

    LO 36: menjelaskan reverse transcriptase PCR (RT-PCR)

    2. Nested PCR

    LO 37: menjelaskan nested-PCR

    Fig. 7.6 Nested


    PCR.
    (a)Standard PCR
    amplification of a
    fragment using a
    primer pair.
    (b)In the second
    PCR, a set of
    primers that lie
    inside the first
    pair is used to
    prime synthesis of
    a shorter
    fragment. Nested
    PCR can be used
    to increase the

    4. RAPD-PCR
    Random amplified polymorphic DNA
    PCR (RAPD-PCR) didasarkan pada
    penempelan primer low-stringency
    RAPD-PCR
    menggunakan
    primer
    tunggal
    RAPD-PCR digunakan untuk
    membuat genomic profiling .
    analisis komparatif
    Kekurangan RAPD-PCR: reproduktibilitas
    rendah,
    yaitu
    kesulitan
    untuk
    memperoleh tingkat penempelan yang
    sama pada percobaan berbeda.

    LO 38: menjelaskan RAPD-PCR

    LO 38: menjelaskan RAPD-PCR

    LO 38: menjelaskan RAPD-PCR

    You might also like