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Genome sequencing

Genome sequencing. Haixu Tang School of Informatics. Methodology for DNA Sequencing. The chain termination method (Sanger et al., 1977) The chemical degradation method (Maxam and Gilbert, 1977) . Chain termination method. Polyacrylamide gel electrophoresis . Sequencing vectors. PCR.

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Genome sequencing

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  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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