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DNA Sequencing

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Modern DNA sequence use PCR technology in sequencing. One primer PCR ... Shotgun Sequencing. Involves fragment assembly using computer algorithms. Contigs ... – PowerPoint PPT presentation

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Title: DNA Sequencing


1
DNA Sequencing
  • Kabi R. Neupane, Ph.D.
  • Leeward Community College
  • ABE Workshop 2006

2
What to Sequence?
  • The RT-PCR product has been inserted into the
    pCR4-TOPO vector
  • Our goal
  • -Sequence the insert DNA

3
We Supplied
Template DNA Your plasmid Primer DNA T3 or T7
4
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

5
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
6
One primer PCR Reaction
DNA Polymerase
Primer
dATP dGTP dCTP dGTP Nucleotides
NEW STRAND
Template DNA
7
Dideoxy Nucleotides
  • Lack an -OH group at the 3-carbon position
  • Cannot add another nucleoside at that position
  • Prevent further DNA synthesis

8
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

9
Chain Termination
10
All Possible Terminations
11
Polyacrylamide Gel Electrophoresis
Separates fragments based on size
12
(No Transcript)
13
DNA Sequence Files
14
Good or Bad?
15
Do not forget the other strand
GGG ATATCACTCA GCATAATTGTTAAGTGACC
T7 Primer
16
  • GenBank
  • The Basic Local Alignment Search Tool (BLAST)
    finds regions of local similarity between
    sequences.

http//www.ncbi.nlm.nih.gov/
17
Shotgun Sequencing
  • Involves fragment assembly using computer
    algorithms

Contigs
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