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    PCR (Polymerase Chain Reaction)

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    Janieka Gan
    PCR (polymerase chain reaction) is a technique used to make millions of copies of a specific DNA sequence. It involves repeated cycles of heating and cooling DNA to separate the strands, allowing primers to attach, and DNA polymerase to synthesize new strands. Key steps include denaturing DNA at high heat, annealing primers at lower heat, and extending new strands during higher heat using DNA polymerase. Repeating these steps exponentially amplifies the target DNA sequence.

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    PCR (Polymerase Chain Reaction)

    Uploaded by

    Janieka Gan
    26 views20 pages
    PCR (polymerase chain reaction) is a technique used to make millions of copies of a specific DNA sequence. It involves repeated cycles of heating and cooling DNA to separate the strands, allowing primers to attach, and DNA polymerase to synthesize new strands. Key steps include denaturing DNA at high heat, annealing primers at lower heat, and extending new strands during higher heat using DNA polymerase. Repeating these steps exponentially amplifies the target DNA sequence.

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    Explanation of Pcr

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    PCR (polymerase chain reaction) is a technique used to make millions of copies of a specific DNA sequence. It involves repeated cycles of heating and cooling DNA to separate the strands, allowing primers to attach, and DNA polymerase to synthesize new strands. Key steps include denaturing DNA at high heat, annealing primers at lower heat, and extending new strands during higher heat using DNA polymerase. Repeating these steps exponentially amplifies the target DNA sequence.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    26 views20 pages

    PCR (Polymerase Chain Reaction)

    Uploaded by

    Janieka Gan
    PCR (polymerase chain reaction) is a technique used to make millions of copies of a specific DNA sequence. It involves repeated cycles of heating and cooling DNA to separate the strands, allowing primers to attach, and DNA polymerase to synthesize new strands. Key steps include denaturing DNA at high heat, annealing primers at lower heat, and extending new strands during higher heat using DNA polymerase. Repeating these steps exponentially amplifies the target DNA sequence.

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    PCR

    (Polymerase
    Chain
    Reaction)

    BY Janieka Gan, Kathleen mago,


    Kimberleigh Metrio
    PCR(Polymerase Chain
    Reaction)
    – What is PCR (polymerase chain reaction)?
    – PCR is a technique used in the lab to make
    millions of copies of a particular section of DNA. It
    was first developed in the 1980s.
    What is PCR?

    – The polymerase chain reaction (PCR) was originally


    developed in 1983 by the American biochemist Kary Mullis.
    He was awarded the Nobel Prize in Chemistry in 1993 for his
    pioneering work.
    – PCR is used in molecular biology to make many copies of
    (amplify) small sections of DNA? or a gene?.
    – Using PCR it is possible to generate thousands to millions of
    copies of a particular section of DNA from a very small
    amount of DNA.
    What is PCR?
    PCR is a common tool used in medical and biological
    research labs. It is used in the early stages of processing
    DNA for sequencing?, for detecting the presence or
    absence of a gene to help identify pathogens ?during
    infection, and when generating forensic DNA profiles from
    tiny samples of DNA.
    How does PCR work?

    – The principles behind every PCR, whatever the sample of


    DNA, are the same.
    – Five core ‘ingredients’ are required to set up a PCR. We
    will explain exactly what each of these do as we go
    along. These are:
    – the DNA template to be copied
    – primers, short stretches of DNA that initiate the PCR
    reaction, designed to bind to either side of the section
    of DNA you want to copy
    How does PCR work?

    – DNA nucleotide bases? (also known as dNTPs). DNA


    bases (A, C, G and T) are the building blocks of DNA and
    are needed to construct the new strand of DNA
    – Taq polymerase enzyme? to add in the new DNA bases
    – buffer to ensure the right conditions for the reaction.
    How does PCR work?

    – PCR involves a process of heating and cooling called thermal


    cycling which is carried out by machine.
    – There are three main stages:
    – Denaturing – when the double-stranded template DNA is
    heated to separate it into two single strands.
    – Annealing – when the temperature is lowered to enable the
    DNA primers to attach to the template DNA.
    – Extending – when the temperature is raised and the new strand
    of DNA is made by the Taq polymerase enzyme.
    How does PCR work?

    – These three stages are repeated 20-40 times, doubling the


    number of DNA copies each time.
    – A complete PCR reaction can be performed in a few hours, or
    even less than an hour with certain high-speed machines.
    – After PCR has been completed, a method called
    electrophoresis can be used to check the quantity and size of
    the DNA fragments produced.
    What happens at each stage of
    PCR?

    – Denaturing stage
    – During this stage the cocktail containing the template DNA and
    all the other core ingredients is heated to 94-95⁰C.
    – The high temperature causes the hydrogen bonds? between the
    bases in two strands of template DNA to break and the two
    strands to separate.
    – This results in two single strands of DNA, which will act as
    templates for the production of the new strands of DNA.
    What happens at each stage of
    PCR?

    – It is important that the temperature is maintained at this


    stage for long enough to ensure that the DNA strands
    have separated completely.
    – This usually takes between 15-30 seconds.
    What happens at each stage of
    PCR?

    – Annealing stage
    – During this stage the reaction is cooled to 50-65⁰C.
    This enables the primers to attach to a specific location on
    the single-stranded template DNA by way of hydrogen
    bonding (the exact temperature depends on the melting
    temperature of the primers you are using).
    – Primers are single strands of DNA or RNA? sequence that are
    around 20 to 30 bases in length.
    What happens at each stage of
    PCR?

    – The primers are designed to be complementary? in sequence


    to short sections of DNA on each end of the sequence to be
    copied.
    – Primers serve as the starting point for DNA synthesis. The
    polymerase enzyme can only add DNA bases to a double
    strand of DNA. Only once the primer has bound can the
    polymerase enzyme attach and start making the new
    complementary strand of DNA from the loose DNA bases.
    What happens at each stage
    of PCR?
    – The two separated strands of DNA are
    complementary and run in opposite directions
    (from one end - the 5’ end – to the other - the 3’
    end); as a result, there are two primers – a
    forward primer and a reverse primer.
    – This step usually takes about 10-30 seconds.
    What happens at each stage
    of PCR?
    – Extending stage
    – During this final step, the heat is increased to 72⁰C to enable
    the new DNA to be made by a special Taq DNA polymerase
    enzyme which adds DNA bases.
    – Taq DNA polymerase is an enzyme taken from the heat-
    loving bacteria? Thermus aquaticus.
    – This bacteria normally lives in hot springs so can tolerate
    temperatures above 80⁰C.
    What happens at each stage
    of PCR?
    – The bacteria's DNA polymerase is very stable at high
    temperatures, which means it can withstand the
    temperatures needed to break the strands of DNA apart
    in the denaturing stage of PCR.
    – DNA polymerase from most other organisms would not
    be able to withstand these high temperatures, for
    example, human polymerase works ideally at 37˚C (body
    temperature).
    What happens at each stage
    of PCR?
    – 72⁰C is the optimum temperature for the Taq
    polymerase to build the complementary strand. It
    attaches to the primer and then adds DNA bases to the
    single strand one-by-one in the 5’ to 3’ direction.
    – The result is a brand new strand of DNA and a double-
    stranded molecule of DNA.
    What happens at each stage
    of PCR?
    – The duration of this step depends on the length of DNA sequence
    being amplified but usually takes around one minute to copy
    1,000 DNA bases (1Kb).
    – These three processes of thermal cycling are repeated 20-40
    times to produce lots of copies of the DNA sequence of interest.
    – The new fragments of DNA that are made during PCR also serve
    as templates to which the DNA polymerase enzyme can attach
    and start making DNA.
    What happens at each stage
    of PCR?
    – The result is a huge number of copies of the specific DNA segment
    produced in a relatively short period of time.
    THANK YOU FOR LISTENING

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