DNA Sequencing

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DNA Sequencing

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

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What to Sequence?

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

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We Supplied
Template DNA Your plasmid Primer DNA T3 or T7
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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

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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
DNA Polymerase
GACTAGTCCT GCAGGTTTAA AGGAATTCGC CCTT
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One primer PCR Reaction
DNA Polymerase
Primer
dATP dGTP dCTP dGTP Nucleotides
NEW STRAND
Template DNA
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Dideoxy Nucleotides

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

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Dideoxy nucleotides

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

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Chain Termination
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All Possible Terminations
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Polyacrylamide Gel Electrophoresis
Separates fragments based on size
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(No Transcript)
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DNA Sequence Files
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Good or Bad?
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Do not forget the other strand
GGG ATATCACTCA GCATAATTGTTAAGTGACC
T7 Primer
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• GenBank
• The Basic Local Alignment Search Tool (BLAST)
  finds regions of local similarity between
  sequences.

http//www.ncbi.nlm.nih.gov/
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Shotgun Sequencing

• Involves fragment assembly using computer
  algorithms

Contigs