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Genome sequencing

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Bacteriophage P1 vectors (Sternberg, 1990) can clone larger fragments of DNA, up ... P1-derived artificial chromosomes or PACs (Ioannou et al., 1994) combine ... – PowerPoint PPT presentation

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Title: Genome sequencing


1
Genome sequencing
  • Haixu Tang
  • School of Informatics

2
Methodology for DNA Sequencing
  • The chain termination method (Sanger et al.,
    1977)
  • The chemical degradation method (Maxam and
    Gilbert, 1977)

3
Chain termination method
4
Polyacrylamide gel electrophoresis
5
Sequencing vectors
6
PCR
7
Sequencing primers
8
Thermal cycle sequencing
9
Automatic DNA sequencing
10
Pyrosequencing
11
Sequencing by hybridization
12
Whole genome shotgun sequencing
13
DNA cloning
14
Clone vectors
  • Bacteriophage P1 vectors (Sternberg, 1990) can
    clone larger fragments of DNA, up to 125 kb using
    current technology.
  • Bacterial artificial chromosomes or BACs (Shizuya
    et al., 1992) can be used to clone fragments of
    300 kb and longer.
  • P1-derived artificial chromosomes or PACs
    (Ioannou et al., 1994) combine features of P1
    vectors and BACs and have a capacity of up to 300
    kb.
  • Fosmids (Kim et al., 1992) contain the F plasmid
    origin of replication and a l cos site. They are
    less prone to instability problems.

15
Assembly of the complete Haemophilus influenzae
genome
16
Problems with the shotgun approach
17
Chromosome walking
18
Chromosome walking by PCR
19
Avoiding errors in WGS
20
Scaffolds
21
Human genome project
  • 300,000 BACs ? sequence-ready map
  • Shotgun sequencing of each BAC
  • Order BACs to get the genome sequences
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