Reverse Genetics and Genome Projects

Reverse Genetics and Genome Projects

1. Reverse Genetics

   • Developed in 1980s
   • Allowed detailed linkage mapping of human genetic disorders using molecular markers
   • Uses positional cloning to identify disease-causing mutations
   • Found links for Neurofibromatosis, Cystic Fibrosis, fragile X-linked mental retardation
   • Laborious process - involves long chromosome walks
     • Chromosome walking (of CF gene)/Positional Cloning
       • Create 2 genomic libraries of the same DNA, cut with different restriction enzymes (EcoR1 and Sal1)
         • 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
       • Unknown gene linked to molecular markers by pedigree analysis
   • Finding the phenotypic function of a DNA sequence

2. Genome Project

   • Radically advanced reverse genetics
   • 1986 - first results from C. elegans genome mapping
     • Discussions about human genome project
     • Work on C. elegans started in 1960s (Brenner)
     • Small differentiated organism
     • Lineage of 959 somatic cells known, 302 neurons mapped
     • Coulson et al, 1986 created a physical map of C. elegans
       • Cosmid libraries (plasmids containing bacterial DNA)
       • Double digest restriction enzyme fingerprinting
       • Computer generated contigs
     • Fingerprinting
       • Cosmid insert digested with Hind III
         • 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
       • Allowed identification of 90-95% of the genome
     • YACs were used after cosmids (linear DNA containing gene being studied - originates from plasmid)
       • Reduced the number of contigs - more overlaps were found
       • Allows larger clones to be made
     • Sulston et al, 1992 - started sequencing
       • 95/100 Mb in contigs <40 gaps
   • Human Genome
     • Genetic linkage map
       • Improved linkage analysis
         • Microsatellite DNA
         • Pedigree collection
         • Software for simultaneous analysis of multiple markers
       • 1416 loci mapped
         • 279 genes, 339 microsatellites
     • Complete contigs (1992)
       • Chromosome 21 (Chumakov et al)
         • Linked to several genetic diseases
       • Y-chromosome (Foote et al)
         • Long arm - heterochromain, variable length (<10 Kb complexity)
         • Short arm - euchromatin, constant length - Y-specific genes and X chromosome homologues
       • YAC Fingerprints (Bellane Chantelot et all)
         • 15-20% of genome in YAC contigs

3. Methods Used in Human Genome Project

   • YAC Fingerprinting
     • YACs digested with restriction enzymes and Southern blotted
       • Hybridized to L1
         • Overlaps found
   • PCR Screening with HC21q STSs
     • 3 YAC libraries from cell lines - heirarchial
     • 180 HC21 specific YAC clones and telomere containing YACs - individually
   • PCR Screening with HCY STSs (sequence-tagged site)
     • YAC library from XYYYY male cell line - heirarchial
     • Individual YACs sized on PFGE
   • By 1995
     • Integrated YAC contigs for chromosome 3,12, 21, 22, 16, Y
     • 83 Mb of cDNA sequence
     • 75% of genome in 225 contigs
     • 21.3 Mb of C. elegans sequence

4. Expressed Sequence Tags (ESTs)

   • Short cDNA sequence
   • Used for gene sequence determination and gene discovery
   • Individual clones from a cDNA library
   • Represent portions of expressed genes
   • ~500-800 nucleotides long
   • 88 000 unique cDNA sequences from 37 human tissues
   • 300 cDNA libraries
     • <20% have no value
     • >50% new genes
   • Overlapped to form THCs (tentative human consensus sequences) - contigs

5. Errors

   • Small sequence gaps
   • Repetitive DNA errors
   • Cloning artefacts

6. Problems with Gene Prediction

   • Introns
   • Alternative splicing
   • Low gene density
   • Trans-splicing
   • 25% of genes are in operons
   • Repeat sequences
     • e.g. can't tell if A and B form a contig with each other or with the same repeat sequence at different locations
     • Combining repeat sequences will cause over collapsing of contig
     • Unitig - contig formed from unique sequences
       • Unitigger - distinguishes between true alignment and alignment due to repeat sequences
         • Prevents overcollapse
         • Based on the coverage that sequence is expected to have, not the number of reads overlapping in the sequence

7. Celera Pipeline

   • Fragments/repeat library
     • Screener produces fragments and marks
       • Overlapper produces fragment overlaps
         • 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
         • Fragment store