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    3 Unit1

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    Rajeev Singh
    This document provides an overview of DNA sequencing and Sanger sequencing. It discusses that sequencing determines the exact order of nucleotides in DNA. Sequencing a genome requires breaking DNA into pieces, sequencing the pieces, and assembling them. Sanger sequencing uses chain termination with dideoxynucleotides which prevents further synthesis, and four reactions with different labeled dideoxynucleotides identify the sequence.

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    3 Unit1

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    Rajeev Singh
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    This document provides an overview of DNA sequencing and Sanger sequencing. It discusses that sequencing determines the exact order of nucleotides in DNA. Sequencing a genome requires breaking DNA into pieces, sequencing the pieces, and assembling them. Sanger sequencing uses chain termination with dideoxynucleotides which prevents further synthesis, and four reactions with different labeled dideoxynucleotides identify the sequence.

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    This document provides an overview of DNA sequencing and Sanger sequencing. It discusses that sequencing determines the exact order of nucleotides in DNA. Sequencing a genome requires breaking DNA into pieces, sequencing the pieces, and assembling them. Sanger sequencing uses chain termination with dideoxynucleotides which prevents further synthesis, and four reactions with different labeled dideoxynucleotides identify the sequence.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    18 views9 pages

    3 Unit1

    Uploaded by

    Rajeev Singh
    This document provides an overview of DNA sequencing and Sanger sequencing. It discusses that sequencing determines the exact order of nucleotides in DNA. Sequencing a genome requires breaking DNA into pieces, sequencing the pieces, and assembling them. Sanger sequencing uses chain termination with dideoxynucleotides which prevents further synthesis, and four reactions with different labeled dideoxynucleotides identify the sequence.

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    18BTC301A: Genomics and Proteomics

    Course Instructor: Dr. Sandhya Sankaran


    What is DNA sequencing

    • Sequencing simply means determining the exact order of the


    bases/nucleotides in a strand of DNA. Because bases exist as pairs,
    and the identity of one of the bases in the pair determines the other
    member of the pair, it is easy to infer sequences in both strands of
    DNA
    • Sequencing an entire genome is sequencing all of the DNA of an
    organism.
    • Complex task- It requires breaking the DNA of the genome into many
    smaller pieces, sequencing the pieces, and assembling the
    sequences/pieces into a single long "consensus."

    2
    Factors that aided sequencing technologies

    • Progress in methods for nucleic acid synthesis


    • The discovery of restriction enzymes
    • The development of gel electrophoresis of nucleic acids

    3
    Sanger sequencing

    • Chain termination method / Dideoxynucleotide


    sequencing

    • Phosphodiester bond cannot form between two adjacent


    nucleotides due to the absence of hydroxyl group in
    ddNTPs. The nucleotide chain cannot synthesis further and
    hence it is known as the chain termination method.

    https://www.youtube.com/watch?v=KTstRrDTmWI
    4
    Steps

    • DNA extraction: Using the proteinase K method or the phenol-


    chloroform DNA extraction method.
    • PCR amplification: Using the flanking primers, dNTPs, ddNTPs, Taq
    DNA polymerase and PCR buffer.
    • Autoradiography, PAGE, or capillary gel electrophoresis- Identification
    of the amplified fragments

    5
    PCR amplification is performed by designing four different reactions

    Reaction PCR reaction Modification

    Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR


    Reaction “A” Labeled ddATPs
    buffer, primers

    Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR


    Reaction “G” Labeled ddGTPs
    buffer, primers

    Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR


    Reaction “T” Labeled ddTTPs
    buffer, primers

    Taq DNA polymerase, dATPs, dGTPs, dCTPs, dGTPs and PCR


    Reaction “C” Labeled ddCTPs
    buffer, primers

    7
    • The flanking primers bind at the region near to the sequence of
    interest.
    • Sequence extension based on the template to varying lengths.

    8
    Summary

    • Class 3 described Sanger sequencing

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