Reverse Genetics and Genome Projects

Reverse Genetics
1. Developed in 1980s
2. Allowed detailed linkage mapping of human genetic disorders using molecular markers
3. Uses positional cloning to identify disease-causing mutations
4. Found links for Neurofibromatosis, Cystic Fibrosis, fragile X-linked mental retardation
5. Laborious process - involves long chromosome walks
  1. Chromosome walking (of CF gene)/Positional Cloning
    1. Create 2 genomic libraries of the same DNA, cut with different restriction enzymes (EcoR1 and Sal1)
      1. Screen one of the libraries (EcoR1) with a probe (MET) linked to the gene in question - find the fragment which binds
        D7S8 is another RFLP marker at the other end of the CF gene
        Digest the fragment with multiple restriction enzymes and separate with electrophoresis to create a restriction map
        Southern blot and mark with MET probe
        Construct a restriction map which shows where MET binds to the fragment
        Sal1 is last binding site nearest to CF gene
        Screen Sal1 digested genomic library with MET
        Find the fragment with Sal1 on either end
        Create a restriction map, as before
        Repeat the process until D7S8 is reached
        Combine all the restriction maps to create a contig map
        Identify which segment contains the CF gene
    2. Unknown gene linked to molecular markers by pedigree analysis
6. Finding the phenotypic function of a DNA sequence
Genome Project
1. Radically advanced reverse genetics
2. 1986 - first results from C. elegans genome mapping
  1. Discussions about human genome project
  2. Work on C. elegans started in 1960s (Brenner)
  3. Small differentiated organism
  4. Lineage of 959 somatic cells known, 302 neurons mapped
  5. Coulson et al, 1986 created a physical map of C. elegans
    1. Cosmid libraries (plasmids containing bacterial DNA)
    2. Double digest restriction enzyme fingerprinting
    3. Computer generated contigs
  6. Fingerprinting
    1. Cosmid insert digested with Hind III
      1. Ends of digests labelled
        Second digest with Sau 3A
        Electrophoresis
        Autoradiography is used to find band sharing of labelled fragments
        Information can be used to create a contig
        Produces many small fragments
    2. Allowed identification of 90-95% of the genome
  7. YACs were used after cosmids (linear DNA containing gene being studied - originates from plasmid)
    1. Reduced the number of contigs - more overlaps were found
    2. Allows larger clones to be made
  8. Sulston et al, 1992 - started sequencing
    1. 95/100 Mb in contigs <40 gaps
3. Human Genome
  1. Genetic linkage map
    1. Improved linkage analysis
      1. Microsatellite DNA
      2. Pedigree collection
      3. Software for simultaneous analysis of multiple markers
    2. 1416 loci mapped
      1. 279 genes, 339 microsatellites
  2. Complete contigs (1992)
    1. Chromosome 21 (Chumakov et al)
      1. Linked to several genetic diseases
    2. Y-chromosome (Foote et al)
      1. Long arm - heterochromain, variable length (<10 Kb complexity)
      2. Short arm - euchromatin, constant length - Y-specific genes and X chromosome homologues
    3. YAC Fingerprints (Bellane Chantelot et all)
      1. 15-20% of genome in YAC contigs
Methods Used in Human Genome Project
1. YAC Fingerprinting
  1. YACs digested with restriction enzymes and Southern blotted
    1. Hybridized to L1
      1. Overlaps found
2. PCR Screening with HC21q STSs
  1. 3 YAC libraries from cell lines - heirarchial
  2. 180 HC21 specific YAC clones and telomere containing YACs - individually
3. PCR Screening with HCY STSs (sequence-tagged site)
  1. YAC library from XYYYY male cell line - heirarchial
  2. Individual YACs sized on PFGE
4. By 1995
  1. Integrated YAC contigs for chromosome 3,12, 21, 22, 16, Y
  2. 83 Mb of cDNA sequence
  3. 75% of genome in 225 contigs
  4. 21.3 Mb of C. elegans sequence
Expressed Sequence Tags (ESTs)
1. Short cDNA sequence
2. Used for gene sequence determination and gene discovery
3. Individual clones from a cDNA library
4. Represent portions of expressed genes
5. ~500-800 nucleotides long
6. 88 000 unique cDNA sequences from 37 human tissues
7. 300 cDNA libraries
  1. <20% have no value
  2. >50% new genes
8. Overlapped to form THCs (tentative human consensus sequences) - contigs
Errors
1. Small sequence gaps
2. Repetitive DNA errors
3. Cloning artefacts
Problems with Gene Prediction
1. Introns
2. Alternative splicing
3. Low gene density
4. Trans-splicing
5. 25% of genes are in operons
6. Repeat sequences
  1. e.g. can't tell if A and B form a contig with each other or with the same repeat sequence at different locations
  2. Combining repeat sequences will cause over collapsing of contig
  3. Unitig - contig formed from unique sequences
    1. Unitigger - distinguishes between true alignment and alignment due to repeat sequences
      1. Prevents overcollapse
      2. Based on the coverage that sequence is expected to have, not the number of reads overlapping in the sequence
Celera Pipeline
1. Fragments/repeat library
  1. Screener produces fragments and marks
    1. Overlapper produces fragment overlaps
      1. Unitigger produces unitigs and overlaps
        Scaffolder produces unitigs, contigs, link bundles and scaffolds
        Consensus sequence
        Assembly and evidence
        Links and distances
        Fragment store
        Fragment store
        Repeat resolver
        Products
      2. Fragment store

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Reverse Genetics and Genome Projects

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	Created by maisie_oj about 11 years ago