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    PCR is a technique used to amplify specific DNA sequences in vitro. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strands are separated by heating, primers anneal to the target sequences, and the polymerase extends the primers to replicate the DNA. This results in exponential amplification of the target DNA sequence. PCR was developed by Kary Mullis in 1983 and has become one of the most widely used techniques in molecular biology due to its ability to rapidly produce millions of copies of a specific DNA fragment.

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    The Basic Principles and Components of PCR (Polymerase Chain Reaction) - Genetics

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    PCR is a technique used to amplify specific DNA sequences in vitro. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strands are separated by heating, primers anneal to the target sequences, and the polymerase extends the primers to replicate the DNA. This results in exponential amplification of the target DNA sequence. PCR was developed by Kary Mullis in 1983 and has become one of the most widely used techniques in molecular biology due to its ability to rapidly produce millions of copies of a specific DNA fragment.

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    © All Rights Reserved
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    8 views29 pages

    Basic Principles and Components of PCR

    Uploaded by

    Joshue Jadie
    PCR is a technique used to amplify specific DNA sequences in vitro. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. During each cycle, the DNA strands are separated by heating, primers anneal to the target sequences, and the polymerase extends the primers to replicate the DNA. This results in exponential amplification of the target DNA sequence. PCR was developed by Kary Mullis in 1983 and has become one of the most widely used techniques in molecular biology due to its ability to rapidly produce millions of copies of a specific DNA fragment.

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    © All Rights Reserved
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    of 29

    Basic Principles and

    Components of PCR

    GENERAL BIOLOGY 2 (FINALS)


    LECTURE 2

    I-5- 1
    I-5- 2
    Ex Vivo / In Vivo / In Vitro / In Situ
    Suppose a sensory neuron is electrically recorded in the
    following settings:
    1. Surgically accessing through the animal's skin, and
    patch-clamped,
    2. Tissue surrounding the neuron is almost separated, but
    still dangling from the larger part of the animal, and
    patch-clamped,
    3. Same as above, but the slight dangling connection is
    cut,
    4. The sensory neuron is isolated and put in an artificially
    oxygenated medium

    I-5- 3
    Development/Invention of PCR Technique

    1993 Nobel Prize in Chemistry


    I-5- 4
    I-5- 5
    REVIEW

    I-5- 6
    PCR: Polymerase Chain Reaction

    n A method of in vitro cloning


    n Allows amplification of specific DNA
    molecules (fragments) in vitro through cycles
    of enzymatic DNA synthesis
    n The most popular and widely used technique
    in all fields of biological studies probably.
    n Why?

    I-5- 7
    I-5- 8
    DNA Replication

    n Purpose: To duplicate DNA molecule


    n Principle:
    q Separation of DNA double-stranded template

    q Primer formation

    q Extension of new DNA strands by a DNA


    polymerase and deoxyribonucleoside
    triphosphates (dNTPs)
    q Other proteins involved

    I-5- 9
    I-5- 10
    I-5- 11
    Principle of PCR

    n Purpose:

    n Requirements:

    n Condition:

    n Components:

    I-5- 12
    Purpose

    n To amplify a lot of double-stranded DNA molecules


    (fragments) with same (identical) size and sequence
    by enzymatic method and cycling condition.

    I-5- 13
    Requirements

    I-5- 14
    I-5- 15
    Condition / Process

    1. Denaturation of DNA template

    2. Annealing of primers

    3. Extension of ds DNA molecules

    I-5- 16
    STEP 1
    Denaturation
    n Melt the barrier between 2 DNA strands
    n Melting temperature (95ºC)
    n Duration: 1-2 mins.
    n Consequences of DNA Strands Separation
    n Decrease in hydrophobic interactions between DNA bases
    n Increase in UV absorbance

    I-5- 17
    I-5- 18
    I-5- 19
    STEP 2
    Annealing

    n Temperature of reaction mixture is cooled to 45-


    60ºC
    n Annealing temperature (50 C)
    n Hydrogen bond REFORM
    n Primers anneal to denatured template DNA

    I-5- 20
    STEP 3
    Extension

    n The temperature is now shifted to 72ºC which is


    ideal for polymerase.
    n Primer are extended by joining the bases
    complementary to DNA strands.
    n Elongation step continues where the polymerase
    adds dNTP's from 5' to 3', reading the template
    from 3' to 5' side, bases are added
    complementary to the template.

    I-5- 21
    Cycling

    Cycle number
    Ramp time

    I-5- 22
    I-5- 23
    I-5- 24
    I-5- 25
    I-5- 26
    Three Aspects of PCR

    I-5- 27
    I-5- 28
    Unusual Origin of PCR, Mullis KB, Scientific American 1990,56

    I-5- 29

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