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    PCR Lecture

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    Pagla Howa
    The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA across several orders of magnitude. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers that flank the target sequence, and dNTPs. Each cycle doubles the amount of target DNA. Key steps in PCR include DNA denaturation to separate the strands, primer annealing, and polymerase extension of the primers to replicate the target. Optimizing factors such as annealing temperature and magnesium concentration is important to maximize amplification efficiency.

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    PCR Lecture

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    Pagla Howa
    154 views18 pages
    The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA across several orders of magnitude. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers that flank the target sequence, and dNTPs. Each cycle doubles the amount of target DNA. Key steps in PCR include DNA denaturation to separate the strands, primer annealing, and polymerase extension of the primers to replicate the target. Optimizing factors such as annealing temperature and magnesium concentration is important to maximize amplification efficiency.

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    The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA across several orders of magnitude. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers that flank the target sequence, and dNTPs. Each cycle doubles the amount of target DNA. Key steps in PCR include DNA denaturation to separate the strands, primer annealing, and polymerase extension of the primers to replicate the target. Optimizing factors such as annealing temperature and magnesium concentration is important to maximize amplification efficiency.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    154 views18 pages

    PCR Lecture

    Uploaded by

    Pagla Howa
    The polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA across several orders of magnitude. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers that flank the target sequence, and dNTPs. Each cycle doubles the amount of target DNA. Key steps in PCR include DNA denaturation to separate the strands, primer annealing, and polymerase extension of the primers to replicate the target. Optimizing factors such as annealing temperature and magnesium concentration is important to maximize amplification efficiency.

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    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 18

    Md. Md. Golam Hossain, Ph.D.

    Pharmacy Discipline Khulna University

    Replication
    DNA

    Transcription
    RNA

    Translation
    PROTEIN

    The central dogma of molecular biology

    Discovery
    PCR was discovered by Kary Mullis in 1983
    On a long motorcycle drive Mentally visualized the process

    Nobel Prize in Chemistry


    1993

    Polymerase Chain Reaction


    Polymerase: DNA polymerase
    DNA polymerase duplicates DNA Before a cell divides, its DNA must be duplicated Chain Reaction: The product of a reaction is used to amplify the same reaction Results in rapid increase in the product

    polymerase chain reaction


    The polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
    PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. including: Selective DNA isolation, Amplification and quantification of DNA, PCR in diagnosis of diseases DNA cloning for sequencing, functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and paternity testing); and the detection and diagnosis of infectious diseases.

    A basic PCR set up requires several components and reagents.[ components include:
    DNA template that contains the DNA region (target) to be amplified. Two primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target. Taq polymerase or another DNA polymerase with a temperature optimum at around 70 C. Deoxynucleoside triphosphates (dNTPs; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand. Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7] Monovalent cation potassium ions.

    primer
    A primer is a strand of nucleic acid that serves as a starting point for DNA synthesis. They are required for DNA replication because the enzymes that catalyze this process, DNA polymerases, can only add new nucleotides to an existing strand of DNA. The polymerase starts replication at the 3'-end of the primer, and copies the opposite strand. In most cases of natural DNA replication, the primer for DNA synthesis and replication is a short strand of RNA

    Thermal Cycling
    A PCR machine controls temperature Typical PCR go through three steps
    Denaturation Annealing Extension

    Denaturation
    Heating separates the double stranded DNA
    Denaturation

    Slow cooling anneals the two strands


    Renaturation

    Heat

    Cool

    Annealing

    Two primers are supplied in molar excess They bind to the complementary region As the DNA cools, they wedge between two template strands Optimal temperature varies based on primer length etc. Typical temperature from 40 to 60 C

    Extension

    DNA polymerase duplicats DNA Optimal temperature 72C

    PCR Amplification
    Exponential Amplification of template DNA

    Typical PCR mix


    In a thin wall Eppendorf tube assemble the following
    PCR components Template DNA (5-200 ng) 1 mM dNTPs (200 uM final) 10 X PCR buffer 25 mM MgCl2 (1.5 mM final) 20 uM forward primer (20 pmoles final) 20 uM reverse primer (20 pmoles final) 5 units/uL Taq DNA polymerase (1.5 units) Water Amount variable 10 uL 5 uL 3 uL 1 uL 1 uL 0.3 uL Variable

    Final Volume

    50 uL

    Steps in PCR
    1. Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. The polymerase binds to the primer-template hybrid and begins DNA synthesis. 2. Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 7580 C, and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute.

    3. Final elongation: This single step is occasionally performed at a temperature of 7074 C for 515 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
    4. Final hold: This step at 415 C for an indefinite time may be employed for short-term storage of the reaction

    Optimising the PCR Reaction Annealing temperature of the primers. The concentration of Mg2+ in the reaction. The extension time. (The denaturing and annealing times.) (The extension temperature.) (The amount of template and polymerasemore is less.)

    Thank you

    Optimising the PCR Reaction Annealing temperature of the primers. The concentration of Mg2+ in the reaction. The extension time. (The denaturing and annealing times.) (The extension temperature.) (The amount of template and polymerase more is less.) 4 Optimising the Annealing Temperature Primers have a calculated annealing temperature (e.g. 54C). Temperature must be confirmed practically. Temperature steps of 2C above and below. Use gradient cycler. Optimising the Mg2+ Concentration The fidelity of the PCR depends on [Mg2+].

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