SangerCoulson Dideoxynucleotide Sequencing

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Sanger-Coulson Dideoxynucleotide Sequencing
Lecture 10/30/00

• Kwamina Bentsi-Barnes
• Deisy Mendoza
• Jennifer Aoki

Best printed in color for clarity
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Topics of Discussion

• Review of last lecture.
• Sequencing.
• Requirements for Sanger-Coulson sequencing.
• Dideoxynucleotides.
• Mechanism of DNA polymerization.
• Sequencing visualization methods.
• Radioactive primer labeled sequencing.
• Radioactive dNTP labeled sequencing.
• Fluorescent primer labeled sequencing.
• Fluorescent ddNTP labeled sequencing.
• Gel separation.
• Gel visualization.
• Gel electrophoresis and readout
• Relative template quantities for sequencing.

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Review of Last Lecture on 10/25/00

• Southern blot
• cDNA probe
• Good vectors
• pGEM -3zf(/-)
• Lambda
• Replicate independently
• Fairly small
• cos-sites wont package, endonucleases recognize
  those sites
• cos site is required for package either in vivo
  or in vitro
• Super Cos
• A lot smaller lambda
• Can replicate individually as a plasmid

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Sequencing

• Sequencing is the process by which you determine
  the exact order of the nucleotides in a given
  region of DNA.
• Dideoxynucleotide sequencing is done through
  complementary chain synthesis and early
  termination.
• The synthesized chains are visualized by methods
  using
• Radioactive labels.
• Nonradioactive labels.

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Requirements for Sanger-Coulson Sequencing

• DNA to be sequenced must be in single strand
  form.
• The region to be sequenced must be 3 flanked by
  known sequence.
• Reagents needed are
• A primer complementary to the known region to
  direct chain synthesis.
• DNA polymerase.
• 4 deoxynucleotide triphosphates (dNTPs).
• 4 dideoxynucleotide triphosphates (ddNTPs).

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Dideoxynucleotides
Here is an example comparing dATP and ddATP
dATP
ddATP
The 3 hydroxyl has been changed to a hydrogen in
ddNTPs, which terminates a DNA chain because a
phosphodiester bond cannot form at this 3
location
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Mechanism of DNA polymerization
Since the 3 OH is changed to a H in ddNTPs,
it is unable to form a phosphodiester bond by
nucleophilic attack on the phosphate, and it will
cause a termination in the DNA chain
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Sequencing Visualization Methods

• Two forms of labeling
• Radioactive
• Primer labeled (32P or 33P)
• dNTP labeled (35S)
• Nonradioactive
• Primer labeled
• ddNTP labeled (big dye terminator)

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Radioactive Primer Labeled Sequencing
Remember each reaction has many molecules each
one incorporating its respective ddNTP and
stopping at a different length.
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Radioactive Deoxynucleotide Labeled Sequencing
What is different about this method? (hint look
at the colors)
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Fluorescent Primer Labeled Sequencing
Whats the big advantage here?
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Fluorescent Dideoxynucleotide Labeled Sequencing
Dont forget that this and the all the previous
reaction vessels have millions of our unknown
fragment. Why do you think were only showing 4
representatives?
Now we run our products on gel
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Gel Separation

• The reaction mixtures are separated on a
  denaturing polyacrylamide gel.
• Denaturing to prevent the DNA from folding up on
  itself while it travels through.
• Polyacrylamide to separate the strands which
  differ in length by only one nucleotide.
• Each band corresponds to a sequence of DNA which
  was terminated by a particular ddNTP.
• This ddNTP is identified by lane in the
  radioactive method and by color in the
  fluorescent method.
• The lowest band on the gel is the shortest. The
  shorter the strand, the earlier in the synthetic
  reaction the ddNTP was incorporated.
• The lowest band on the gel is at the 5 end of
  our synthesized strand and is complementary to
  the 3 end of our unknown fragment.

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Gel Visualization

• Radioactive method which requires four gel lanes,
  one for each reaction vessel.
• Readout is done by hand or with a densitometric
  scanner.
• Nonradioactive fluorescence sequencing requires
  only one gel lane because each nucleotide has a
  distinct color.
• The readout process is done by laser scanner and
  recorded by computer.

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Gel Electrophoresis and Readout of Reaction
Products
vs.
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Relative Template Quantities needed for Sequencing

• Most
• Double stranded plasmid (must denature)
  standard sequencing reaction
• Each molecule of template used only once

• Less
• Single stranded construct such as phagemid or M13
  standard sequencing
• Each molecule of template used only once

• Least
• Either double stranded or single stranded
  construct types or fragments cycle sequencing
• Each molecule is used as template up to 30 times

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That's all Folks
Hope you learned something