Scribd Logo
Upload

Welcome to Scribd!

  • 82 views

    Lecture 36 Sequencing

    Uploaded by

    Jenna Scheibler
    DNA sequencing involves determining the order of nucleotide bases in DNA. There are two main methods - biochemical degradation and enzymatic synthesis. The most commonly used method is the Sanger method of enzymatic synthesis, which relies on DNA chain termination by dideoxynucleotide triphosphates to generate DNA fragments of different lengths that can be resolved by electrophoresis to determine the nucleotide sequence. Automation and fluorescent dyes have improved the Sanger method, allowing for high-throughput sequencing using capillary electrophoresis.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd

    Lecture 36 Sequencing

    Uploaded by

    Jenna Scheibler
    82 views12 pages
    DNA sequencing involves determining the order of nucleotide bases in DNA. There are two main methods - biochemical degradation and enzymatic synthesis. The most commonly used method is the Sanger method of enzymatic synthesis, which relies on DNA chain termination by dideoxynucleotide triphosphates to generate DNA fragments of different lengths that can be resolved by electrophoresis to determine the nucleotide sequence. Automation and fluorescent dyes have improved the Sanger method, allowing for high-throughput sequencing using capillary electrophoresis.

    Original Description:

    FS 362 PURDUE UNIVERSITY

    Copyright

    © Attribution Non-Commercial (BY-NC)

    Available Formats

    PDF, TXT or read online from Scribd

    Did you find this document useful?

    Is this content inappropriate?

    DNA sequencing involves determining the order of nucleotide bases in DNA. There are two main methods - biochemical degradation and enzymatic synthesis. The most commonly used method is the Sanger method of enzymatic synthesis, which relies on DNA chain termination by dideoxynucleotide triphosphates to generate DNA fragments of different lengths that can be resolved by electrophoresis to determine the nucleotide sequence. Automation and fluorescent dyes have improved the Sanger method, allowing for high-throughput sequencing using capillary electrophoresis.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    82 views12 pages

    Lecture 36 Sequencing

    Uploaded by

    Jenna Scheibler
    DNA sequencing involves determining the order of nucleotide bases in DNA. There are two main methods - biochemical degradation and enzymatic synthesis. The most commonly used method is the Sanger method of enzymatic synthesis, which relies on DNA chain termination by dideoxynucleotide triphosphates to generate DNA fragments of different lengths that can be resolved by electrophoresis to determine the nucleotide sequence. Automation and fluorescent dyes have improved the Sanger method, allowing for high-throughput sequencing using capillary electrophoresis.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    You are on page 1of 12

    DNA Sequencing

    DNA sequencing
    Biochemical process to determine of the order of nucleotide bases
    Chemical degradation
    (Maxam and Gilbert, 1977)

    Enzymatic synthesis
    Sanger Method (Sanger et al., 1977)

    Generate sequences that begin at fixed point and terminate at a particular type of residue (A, T, G or C for Sanger Method)

    DNA sequencing
    Sanger method has served as the workhorse of DNA sequencing projects for the past 30 years
    Human genome sequencing project >900 microbial genome sequences available Multiple genome sequences available for many foodborne pathogens
    >20 genome sequences available for Listeria monocytogenes

    DNA sequencing
    Sequencing relies on DNA chain termination by dideoxynucleotide triphosphates (ddNTPs)

    Cycle sequencing demonstration

    DNA sequencing
    Termination points are nucleotide specific but occur randomly along length of target DNA Generates many fragments of different lengths Fragments resolved by electrophoresis
    Discriminate DNAs that differ in length by as little as a single nucleotide Four populations loaded into adjacent lanes of sequencing gel, order of nucleotides along DNA can be read from gel image

    Classic Sanger sequencing


    Sequencing performed in four separate reactions each containing one of the four ddNTPs Run on adjacent lanes in a denaturing polyacrylamide gel Short runs and can be difficult to interpret

    Automation of Sanger Method


    Cycle sequencing/Dye-terminator 15-40 rounds of conventional thermal cycling
    Denaturation of double stranded DNA Annealing of primer to sequence Extension of primer by thermal stable DNA polymerase Termination of extended strand by ddNTP incorporation

    Fluorescent dyes to ddNTPs that become incorporated to 3 end of sequencing reaction products
    Same primer in a single tube

    Capillary electrophoresis

    Electropherograms

    Key to consistent successful cycle sequencing is cleanliness of DNA template


    Impurities (e.g., agarose, salts, proteins) cause premature termination and pausing of DNA polymerase Degraded DNA cause false priming

    DNA sequencing success

    DNA template should be purified and the quantity and quality of DNA template should be assessed prior to submitting samples for sequencing

    Illumina Sequencing by Synthesis


    See link to PDF blackboard

    Ion Torrent (Personal Genome Maching (PGM))


    http://www.iontorrent.com/thesimplest-sequencing-chemistry/

    You might also like

    pFad - Phonifier reborn

    Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

    Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


    Alternative Proxies:

    Alternative Proxy

    pFad Proxy

    pFad v3 Proxy

    pFad v4 Proxy