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    Gaurav jain
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    PCR involves copying DNA sequences using primers, DNA polymerase, and cycling between high and low temperatures. The process includes: 1) Melting the DNA strands at a high temperature. 2) Allowing primers to anneal to the single-stranded DNA at a lower temperature. 3) Using DNA polymerase to synthesize new strands complementary to the templates at an intermediate temperature. This cycling is repeated for 30 or more cycles, exponentially amplifying the target DNA region.

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    PCR involves copying DNA sequences using primers, DNA polymerase, and cycling between high and low temperatures. The process includes: 1) Melting the DNA strands at a high temperature. 2) Allowing primers to anneal to the single-stranded DNA at a lower temperature. 3) Using DNA polymerase to synthesize new strands complementary to the templates at an intermediate temperature. This cycling is repeated for 30 or more cycles, exponentially amplifying the target DNA region.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    5 views13 pages

    CorrectPrimer PCRslides

    Uploaded by

    Gaurav jain
    PCR involves copying DNA sequences using primers, DNA polymerase, and cycling between high and low temperatures. The process includes: 1) Melting the DNA strands at a high temperature. 2) Allowing primers to anneal to the single-stranded DNA at a lower temperature. 3) Using DNA polymerase to synthesize new strands complementary to the templates at an intermediate temperature. This cycling is repeated for 30 or more cycles, exponentially amplifying the target DNA region.

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    of 13

    Polymerase Chain Reaction (PCR)

    Steven Salzberg
    How do we make copies of DNA?
    Recall that DNA sticks to itself (“hybridizes”)

    5’ - ACACCGGTTCGTAGAGCAT - 3’
    3’ - TGTGGCCAAGCATCTCGTA - 5’
    Primers

    5'-CTATTCATTCAATCATACACCCAA
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3'
    CTAAGAAACACCACCCAGAC-5'

    Here we have 2 primers, one in green, one in blue


    Melt (heat up gently)

    5'-CTATTCATTCAATCATACACCCAA

    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3'

    CTAAGAAACACCACCCAGAC-5'
    Anneal (cool down gently)

    5'-CTATTCATTCAATCATACACCCAA
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3'
    CTAAGAAACACCACCCAGAC-5'
    Add copier molecule: DNA polymerase

    5'-CTATTCATTCAATCATACACCCAA
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3'
    CTAAGAAACACCACCCAGAC-5'
    Result after round one:

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3'
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3’
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'
    Result after round two:
    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3’
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3’
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3’
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'

    5'-CTATTCATTCAATCATACACCCAAGATAGATAGATAGATAGATAGATAGATAGATAGATTCTTTGTGGTGGGTCTG-3’
    3'-GATAAGTAAGTTAGTATGTGGGTTCTATCTATCTATCTATCTATCTATCTATCTATCTAAGAAACACCACCCAGAC-5'
    Repeat for 30 cycles or more...
    Summary: the PCR recipe
    Ingredients
    ● DNA
    ● primers
    ● DNA polymerase
    ● A’s, C’s, G’s, T’s
    Summary: the PCR recipe
    1. Melt at 94o C
    2. Cool to 54o C
    3. Warm to 72o C
    4. Go to step 1

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