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    Dna Sequencing by Sanger Method

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    Sethu SK
    This document describes the Sanger sequencing method, including the materials and setup used. It explains how DNA replication occurs in the presence of both normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs), generating DNA fragments of different lengths. An example is provided of running a DNA sample with one of the nucleotide mixtures, showing the resulting band pattern on a gel, and asks what the band patterns from the other mixtures would look like.

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    Dna Sequencing by Sanger Method

    Uploaded by

    Sethu SK
    85 views19 pages
    This document describes the Sanger sequencing method, including the materials and setup used. It explains how DNA replication occurs in the presence of both normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs), generating DNA fragments of different lengths. An example is provided of running a DNA sample with one of the nucleotide mixtures, showing the resulting band pattern on a gel, and asks what the band patterns from the other mixtures would look like.

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    very good for sequencing

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    This document describes the Sanger sequencing method, including the materials and setup used. It explains how DNA replication occurs in the presence of both normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs), generating DNA fragments of different lengths. An example is provided of running a DNA sample with one of the nucleotide mixtures, showing the resulting band pattern on a gel, and asks what the band patterns from the other mixtures would look like.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    85 views19 pages

    Dna Sequencing by Sanger Method

    Uploaded by

    Sethu SK
    This document describes the Sanger sequencing method, including the materials and setup used. It explains how DNA replication occurs in the presence of both normal deoxynucleotide triphosphates (dNTPs) and chain-terminating dideoxynucleotide triphosphates (ddNTPs), generating DNA fragments of different lengths. An example is provided of running a DNA sample with one of the nucleotide mixtures, showing the resulting band pattern on a gel, and asks what the band patterns from the other mixtures would look like.

    Copyright:

    © All Rights Reserved
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    of 19

    Introduction

    to
    Bioinformatics
    BIN101

    DNA Sequencing by Sangers Method


    Sanger Sequencing Materials
     DNA Polymerase
     Primer (radioactive)
     Template DNA
     dNTPs (deoxyNTP)
     ddNTPs
    (dideoxyNTP)
    Sanger Sequencing Setup

     What do you think will happen if you allow DNA


    replication to occur in the presence of both
    deoxyATP and dideoxyATP?

    Tube A
    15% ATP
    25% GTP
    25% TTP
    25% CTP
    10% ddATP
    Sanger Sequencing Setup

    AAT C T G G G C TA C T C G G G C G T T
    C G CA

    Tube A
    15% ATP
    25% GTP
    25% TTP
    25% CTP
    10% ddATP
    Sanger
    Sequencing
    AAT C T G G G C TA C T C G G G C G T T
    C G CA

    Setup A G C C C G CA
    Tube A
    15% ATP
    25% GTP
    25% TTP
    25% CTP
    10% ddATP
    Sanger
    Sequencing
    AAT C T G G G C TA C T C G G G C G T T
    C G CA

    Setup A G C C C G CA

    AT G A G C C C G CA
    Tube A
    15% ATP
    25% GTP
    25% TTP
    25% CTP
    10% ddATP
    Sanger
    Sequencing
    AAT C T G G G C TA C T C G G G C G T T
    C G CA

    Setup A G C C C G CA

    AT G A G C C C G CA
    Tube A
    15% ATP
    25% GTP
    AC C C GATGAG C C C
    GGCC
    GAT
    25% TTP
    25% CTP
    10% ddATP
    Sanger
    Sequencing
    AAT C T G G G C TA C T C G G G C G T T
    C G CA

    Setup A G C C C G CA

    AT G A G C C C G CA
    Tube A
    15% ATP
    25% GTP
    AC C C GATGAG C C C
    GGCC
    GAT
    25% TTP
    A G A C C C G A T G A G C C C G CA 25% CTP
    10% ddATP
    Problem:
    1. The following DNA fragment was sequenced by the Sanger method. The red asterisk
    indicates a fluorescent label.

    A sample of the DNA was reacted with DNA polymerase and each of the nucleotide
    mixtures (in an appropriate buffer) listed below. Dideoxynucleotides (ddNTPs) were added
    in relatively small amounts.
    1. dATP, dTTP, dCTP, dGTP, ddTTP
    2. dATP, dTTP, dCTP, dGTP, ddGTP
    3. dATP, dCTP, dGTP, ddTTP
    4. dATP, dTTP, dCTP, dGTP
    The resulting DNA was separated by electrophoresis on an agarose gel, and the fluorescent
    bands on the gel were located. The band pattern resulting from nucleotide mixture
    1 is shown below.
    Assuming that all mixtures were
    run on the same gel,
    what did the remaining lanes
    of the gel look like?

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