DNA Sequencing: The Chain Termination Method (Sanger Method)

00:03:00
https://www.youtube.com/watch?v=vK-HlMaitnE

Zusammenfassung

TLDRThe Sanger method, or chain termination method, is a classic DNA sequencing technique that involves several key steps. First, double-stranded DNA is denatured into single strands. A primer is then attached to one strand, followed by adding DNA polymerase, regular nucleotides, and specially labeled nucleotides (ddNTPs) for termination. After reaction mixtures are prepared, the DNA strands are separated by size through gel electrophoresis. The final bands, visible on X-ray film, indicate the DNA sequence by matching the labeled nucleotide at the end of each fragment.

Mitbringsel

  • 🧬 The Sanger method is also known as chain termination sequencing.
  • 🔬 DNA is denatured with heat to form single strands.
  • 📏 Gel electrophoresis separates DNA fragments by size.
  • 🔍 Labeled ddNTPs allow for sequence determination through X-ray.
  • 🔗 All strands begin with the same primer for uniformity.
  • 🔢 Single nucleotide differences can be resolved on the gel.

Zeitleiste

  • 00:00:00 - 00:03:00

    This video introduces the Sanger method, also known as the chain termination method for DNA sequencing. The process begins by denaturing double-stranded DNA into single strands using heat, resulting in a template strand and a complementary strand. A primer is attached to the template strand to enable nucleotide addition. Four reaction mixtures are prepared, each containing the template DNA and primer, along with free nucleotides, some of which are radiolabeled for tracking. Modified nucleotides (ddNTPs) are added to each mixture, with each mixture containing only one type of ddNTP, causing chain termination when a ddNTP is incorporated. The DNA fragments are then separated by size using gel electrophoresis. The smaller fragments migrate faster towards the positive electrode, and after drying the gel, X-ray films reveal bands indicative of the DNA sequence. Each reaction mixture starts with the same primer but ends with different sequences corresponding to the ddNTPs added, allowing determination of the DNA sequence by analyzing the bands on the X-ray film.

Mind Map

Video-Fragen und Antworten

  • What is the Sanger method of DNA sequencing?

    The Sanger method, also known as the chain termination method, is a well-tested technique for sequencing DNA.

  • How is DNA prepared for sequencing?

    DNA is denatured by applying heat to convert it from double-stranded to single-stranded.

  • What role do primers play in the Sanger method?

    Primers anneal to the template strand to allow the addition of nucleotides.

  • What are ddNTPs?

    Modified nucleotides (ddNTPs) are added to the reaction mixtures and cause chain termination.

  • How is the DNA separated by size?

    DNA is separated using gel electrophoresis, which differentiates DNA fragments by size.

  • How is the sequencing read from the results?

    The sequence can be determined by reading the bands formed on the X-ray film after electrophoresis.

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Automatisches Blättern:
  • 00:00:00
    welcome to this video animation on the
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    sander method of DNA sequencing this
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    method is also known as the chain
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    termination method of DNA sequencing
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    this is a well tested method of
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    sequencing and the following video will
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    atline the principles on which this
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    method of DNA sequencing
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    Works firstly the DNA to be sequenced
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    must be denatured and converted from
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    double stranded DNA into single stranded
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    DNA this is done through the application
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    of heat the DNA splits into a template
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    Strand and it's complimentary
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    strand a primer is then analed to the
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    template strand this is to allow the
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    addition of nucleotides later four
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    reaction mixtures are set up added to
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    each of these is the tempate stranded
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    DNA with attached primer this is the DNA
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    that will be
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    sequenced this is followed by the
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    addition of DNA
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    polymeris free nucleotides are DNA NPS
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    are then added to the reaction mixtures
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    one of these dntps is usually radiol
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    labeled with a 32 phosphorus or 35
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    sulfur atom this helps in determining
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    the DNA sequence later modified
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    nucleotides or DD ntps are then added to
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    the reaction mixtures only one type of
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    DD ntp is added to each reaction mixture
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    chain termination occurs after the
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    addition of ddntp due to the fact that
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    there's there's no o group available to
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    attack the next dntp the DNA must now be
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    separated on size this is done using a
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    process known as gel
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    electroforesis a sample of each of the
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    four reaction mixtures is taken and
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    pipetted into a separate Lane on the gel
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    polyacrylite gel electroforesis can
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    separate polynucleotide chains differing
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    in size by one
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    nucleotide a current is set up across
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    the gel and the DNA is inserted at the
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    end with the negative elect
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    as the DNA is negatively charged it will
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    begin to migrate towards the positive
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    electrode smaller fragments will move
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    faster than larger fragments the gel is
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    dried down and an x-ray taken since we
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    radio labeled at dntp we can now see it
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    show up on the X-ray film it will show
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    up as bands across the X-ray film which
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    can be used to find out the sequence of
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    DNA we know that each reaction mixture
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    had the same primer therefore all of the
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    strands begin with the same sequence
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    each chain in a particular flask ends in
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    a sequence determined by whatever dioxy
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    ntp was added to that flask for example
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    a DNA strand taken from the flask to
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    which dioxy ATP was added will end in an
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    A or added nucleotide we can use this
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    information to determine the sequence of
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    DNA by reading across the various bands
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    on the X-ray fil
Tags
  • Sanger method
  • DNA sequencing
  • chain termination
  • gel electrophoresis
  • ddNTPs
  • nucleotide
  • primer
  • denaturation
  • X-ray film
  • template strand