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    DNA Sequencing: Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

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    ximenalr
    The document provides an overview of DNA sequencing. It discusses that the goal is to sequence the insert DNA that has been inserted into a plasmid vector using primers. It describes Frederick Sanger's discovery of chain termination sequencing and use of DNA polymerase. The method exploits DNA polymerase activity and uses dideoxynucleotides which lack a 3'-OH group, preventing further DNA synthesis and resulting in terminated strands of different lengths that can be separated on a polyacrylamide gel to determine the DNA sequence. Sequence files can be submitted to GenBank and regions of similarity identified using BLAST. Shotgun sequencing involves fragment assembly using computer algorithms to construct contigs.

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    DNA Sequencing: Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

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    ximenalr
    51 views17 pages
    The document provides an overview of DNA sequencing. It discusses that the goal is to sequence the insert DNA that has been inserted into a plasmid vector using primers. It describes Frederick Sanger's discovery of chain termination sequencing and use of DNA polymerase. The method exploits DNA polymerase activity and uses dideoxynucleotides which lack a 3'-OH group, preventing further DNA synthesis and resulting in terminated strands of different lengths that can be separated on a polyacrylamide gel to determine the DNA sequence. Sequence files can be submitted to GenBank and regions of similarity identified using BLAST. Shotgun sequencing involves fragment assembly using computer algorithms to construct contigs.

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    DNA Sequencing

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    The document provides an overview of DNA sequencing. It discusses that the goal is to sequence the insert DNA that has been inserted into a plasmid vector using primers. It describes Frederick Sanger's discovery of chain termination sequencing and use of DNA polymerase. The method exploits DNA polymerase activity and uses dideoxynucleotides which lack a 3'-OH group, preventing further DNA synthesis and resulting in terminated strands of different lengths that can be separated on a polyacrylamide gel to determine the DNA sequence. Sequence files can be submitted to GenBank and regions of similarity identified using BLAST. Shotgun sequencing involves fragment assembly using computer algorithms to construct contigs.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Flag for inappropriate content
    Download as ppt, pdf, or txt
    51 views17 pages

    DNA Sequencing: Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

    Uploaded by

    ximenalr
    The document provides an overview of DNA sequencing. It discusses that the goal is to sequence the insert DNA that has been inserted into a plasmid vector using primers. It describes Frederick Sanger's discovery of chain termination sequencing and use of DNA polymerase. The method exploits DNA polymerase activity and uses dideoxynucleotides which lack a 3'-OH group, preventing further DNA synthesis and resulting in terminated strands of different lengths that can be separated on a polyacrylamide gel to determine the DNA sequence. Sequence files can be submitted to GenBank and regions of similarity identified using BLAST. Shotgun sequencing involves fragment assembly using computer algorithms to construct contigs.

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    Download as PPT, PDF, TXT or read online from Scribd
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    DNA Sequencing

    Kabi R. Neupane, Ph.D. Leeward Community College ABE Workshop 2006

    What to Sequence?

    The RT-PCR product has been inserted into the pCR4-TOPO vector Our goal -Sequence the insert DNA

    We Supplied

    Template DNA: Your plasmid Primer DNA: T3 or T7

    Frederick Sanger
    Discovered DNA sequencing by chain termination method Nobel Prize 1 (1958)
    Complete amino acid sequence of insulin

    Nobel Prize 2 (1980)


    For DNA sequencing

    DNA Polymerase Action


    DNA Sequencing exploits the DNA polymerase activity for deciphering DNA sequence Modern DNA sequence use PCR technology in sequencing T3 Primer
    ATTAACCC TCACTAAAGG

    GACTAGTCCT GCAGGTTTAA

    DNA AGGAATTCGC CCTT Polymerase

    One primer PCR Reaction


    DNA Polymerase Primer
    dATP dGTP dCTP dGTP Nucleotides

    NEW STRAND

    Template DNA

    Dideoxy Nucleotides
    Lack an -OH group at the 3-carbon position Cannot add another nucleoside at that position Prevent further DNA synthesis

    Dideoxy nucleotides
    Incorporation of a dideoxynucleotide to growing DNA strand terminates its further extension Are added in small proportion
    dATP dGTP dCTP dTTP ddATP ddGTP ddCTP ddTTP

    Chain Termination

    All Possible Terminations

    Polyacrylamide Gel Electrophoresis

    Separates fragments based on size

    DNA Sequence Files

    Good or Bad?

    Do not forget the other strand

    GGG ATATCACTCA GCATAATTGTTAAGTGACC

    T7 Primer

    GenBank The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences.

    http://www.ncbi.nlm.nih.gov/

    Shotgun Sequencing
    Involves fragment assembly using computer algorithms

    Contigs

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