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    Polymerase Chain Reaction

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    GENESIS sHINE
    PCR was developed in 1983 by Kary Mullis and has revolutionized molecular biology. It allows for the in vitro amplification of specific DNA sequences, enabling scientists to generate millions of copies of a particular DNA segment. The key components of PCR are target DNA, two primers, a thermostable DNA polymerase, dNTPs, and a buffer solution. During each cycle of PCR, the target DNA is denatured, primers anneal, and new DNA strands are extended. This allows exponential amplification of the target sequence. Since its discovery, PCR has enabled advances in fields such as forensics, genetics, medicine, and molecular engineering.

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    Polymerase Chain Reaction

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    GENESIS sHINE
    59 views16 pages
    PCR was developed in 1983 by Kary Mullis and has revolutionized molecular biology. It allows for the in vitro amplification of specific DNA sequences, enabling scientists to generate millions of copies of a particular DNA segment. The key components of PCR are target DNA, two primers, a thermostable DNA polymerase, dNTPs, and a buffer solution. During each cycle of PCR, the target DNA is denatured, primers anneal, and new DNA strands are extended. This allows exponential amplification of the target sequence. Since its discovery, PCR has enabled advances in fields such as forensics, genetics, medicine, and molecular engineering.

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    PCR was developed in 1983 by Kary Mullis and has revolutionized molecular biology. It allows for the in vitro amplification of specific DNA sequences, enabling scientists to generate millions of copies of a particular DNA segment. The key components of PCR are target DNA, two primers, a thermostable DNA polymerase, dNTPs, and a buffer solution. During each cycle of PCR, the target DNA is denatured, primers anneal, and new DNA strands are extended. This allows exponential amplification of the target sequence. Since its discovery, PCR has enabled advances in fields such as forensics, genetics, medicine, and molecular engineering.

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    © All Rights Reserved
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    Download as pptx, pdf, or txt
    59 views16 pages

    Polymerase Chain Reaction

    Uploaded by

    GENESIS sHINE
    PCR was developed in 1983 by Kary Mullis and has revolutionized molecular biology. It allows for the in vitro amplification of specific DNA sequences, enabling scientists to generate millions of copies of a particular DNA segment. The key components of PCR are target DNA, two primers, a thermostable DNA polymerase, dNTPs, and a buffer solution. During each cycle of PCR, the target DNA is denatured, primers anneal, and new DNA strands are extended. This allows exponential amplification of the target sequence. Since its discovery, PCR has enabled advances in fields such as forensics, genetics, medicine, and molecular engineering.

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    of 16

    SUPERVISED BY- PRESENTED BY-

    PROF. S. P. VYAS SUDIPTA NANDI


    M.PHARM 1ST SEMESTER
    ROLL NO- Y19254029

    Department of Pharmaceutical Sciences


    Dr. Harisingh Gour Vishwavidyalaya
    (A Central University)
    Sagar, M.P.
    TOPICS TO BE DEALT WITH
     WHAT IS PCR
     HISTORY AND PERSPECTIVE OF PCR
     COMPONENTS OF PCR
     PRINCIPLES INVOLVED
     TECHNIQUE OF PCR
     INSTRUMENTATION
     ADVANTAGES
     APPLICATIONS
     VARIANTS OF PCR
     FUTURE PROSPECTIVES
     REFERENCES
    2
    WHAT IS PCR

     PCR is laboratory or in vitro cell-free


    amplification technique
     Uses specific sequence of DNA of small
    amount and amplify to synthesize multiple
    identical copies of DNA segment of interest.

    3
    From its birth, PCR changed the molecular biology world.

    • Dr. Kary Mullis developed PCR


    1983

    • First publication of PCR by Cetus Corporation appears in Science


    1985

    • Purified Taq polymerase was first used in PCR


    1986

    • PerkinElmer introduced the automated thermal cycler


    1988

    • Taq polymerase declared as “Molecule of the year”


    1989

    • Dr. Kary Mullis shares Nobel Prize in Chemistry for conceiving PCR
    1993 technology

    4
    COMPONENTS OF PCR
    • Essential requirements include :
     Target DNA : 0.1- 10 kbp in length; upto 40 kbp.
     Two DNA primers (synthetic oligonucleotide of 17-30 nucleotides length; 0.1-
    0.5μM)
     A thermostable DNA polymerase (e.g. Taq polymerase; 1- 2.5 units)
     Four deoxyribonucleotides (dATP, dCTP, dGTP, dTTP; 20- 200 μM)
     Buffer : pH 8.3- 8.8
     Bivalent cations (Mg2+, Mn2+)

    5
    PRINCIPLE INVOLVED

    1. Separation of DNA double-stranded template

    2. Hybridization of strand with primer

    3. Extension of new DNA strands by a DNA polymerase


    and deoxynucleoside triphosphates (dNTPs)

    6
    TECHNIQUE OF PCR
    • PCR consists of a series of 20- 40 repeated temperature changes called
    thermal cycles, with each cycle commonly consists of 2-3 discrete
    temperature steps as below:

    DENATURATION
    ANNEALING
    of ds DNA
    of primers
    template

    SYNTHESIS of ds
    DNA molecule

    7
    TECHNIQUE OF PCR
     DENATURATION :
    Samples are heated to 94-96°C for 30 sec
    to 1 minute to denature the two strands
    of of ds target DNA.

     RENATURATION OR ANNEALING :
    Next the temperature is slowly
    cooled to 55-65°C for 30 sec or
    more, the primers base pair with the
    complementary regions flanking
    target DNA.

     EXTENSION OR SYNTHESIS :
    Finally the temperature is raised to 75-
    80°C, allows the Taq polymerase to bind
    at each primed site and extend a new
    DNA strand.

    8
    AT A GLIMPSE….

    9
    INSTRUMENTATION
     Quantitative PCR instrument is a machine that amplifies and detects DNA.
     It combines the functions of
     Thermal cycler
     fluorimeter
    ADVANTAGES
     Sensitivity
     Speed
     Easy and simple
     Cost effective
     flexibility
     Usually not necessary to use radioactive
    materials
     More precise in determining size of alleles

    11
    APPLICATIONS

    Molecular Identification Sequencing Genetic Engineering


     Molecular Archaeology  Bioinformatics  Site-directed
     Molecular Epidemiology  Genomic Cloning mutagenesis
     Molecular Ecology  Human Genome  Gene Expression
     DNA fingerprinting Project (HGP) Studies
     Classification of
    organisms PRENATAL DIAGNOSIS
     Genotyping FORENSIC LABORATORY
     Mutation screening
     Drug discovery
     Genetic matching
     Detection of pathogens
     Diagnostic purpose

    12
     Inverse PCR
     In-situ PCR
     Anchored PCR
     Reverse Transcription PCR  Asymmetric PCR
    (RT-PCR)  Assembly PCR
     Real-time PCR  Hot-start PCR
     Miniprime PCR  Touchdown PCR
     Multiplex PCR  Solid phase PCR
     Variable Number of
    Tandem Repeats (VNTR)  Intersequence Specific PCR
    (ISSR)
     Quantitative real time PCR
    (Q- RT PCR)  Suicide PCR
     Nested PCR  Colony PCR
     Long-range PCR  Digital PCR
     Single cell PCR  Ligation mediated PCR
     High-fidelity PCR
     Cold PCR

    13
    FUTURE PROSPECTIVES
     Preimplantation diagnosis
     PCR and Virology
     PCR and Bacteriology
     Cancer therapy

     CONTINUOUS FLOW PCR (The future of PCR) –


    Most interesting and innovative way to increase speed of
    PCR.
    • Advantage :
     Reduce time needed for reaction
     Thermal equilibrium can be reached easily

     In an recent approach (February, 2019) Bio-Rad released


    first FDA-cleared droplet digital PCR system and BCR-ABL
    fusion test for monitoring chronic myeloid leukemia
    treatment response.

    14
     Satyanarayana U, Chakrapani U. “Biochemistry”, Polymerase Chain
    Reaction, Books & allied (P) Ltd.,2013, 4th edition;594-596.
     Vyas S. P, Dixit V. K, “Pharmaceutical Biotechnology”, CBS Publishers,
    New Delhi,1998, 1st edition;353
     Innis Michael A., Gelfand David h., Sninsky John j., “PCR
    APPLICATIONS- PROTOCOLS FOR FUNCTIONAL GENOMICS”,
    Academic press,1999;33-263.
     Mullis Kary B, Ferre Francois, Gibbs Richard A. “PCR – The Polymerase
    Chain Reaction”, Springer Science+Business Media, New York, 1994;165-
    182.

    15
    “….innovation is also on its face often marked with a bit of insanity.
    If no one mentions loudly that one things you’re out of your mind,
    then, you probably not being innovative.”
    - Kary B. Mullis

    THANK
    YOU

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