For the next few weeks:

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For the next few weeks

• Genome projects
• Chapter 4
• DNA fingerprinting
• Chapter 10
• Cloning and genetically modified organisms
• Chapter 21, Strachan (will be handed out)
• Ethical considerations of human genetics
• Genome, Matt Ridley
• Grad student presentations

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Genome projects

• E. coli - 4.6 Mb - 1997
• S. cerevisiae - 12 Mb - 1996 - 6,000 genes
• C. elegans - 100 Mb - 1998 - 20,000 genes
• D. melanogaster - 165 Mb - 2000 - 13,000 genes
• Arabidopsis - 2001
• Fugu (pufferfish) - 400 Mb
• Mouse - 3000 Mb
• Human - 3000 Mb - 2001 - 65,000- 80,000 genes
  estimated

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History of genome project

• First formal proposal to sequence genome in 1985
• 1990 - Human genome project (HGP) initiated in US
  with 15 year plan for completing genome
• Human Genome Organization (HUGO) founded to
  coordinate international efforts
• 1998 - Celera Genomics announced their 3 year
  plan to sequence genome
• TIGR (The Institute for Genomic Research)
• Developed technology to characterize and annotate
  ESTs

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Genome project timeline
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Goals of project

• Genetic map
• Physical map
• Develop DNA sequence technology
• Gene identification
• Technology development
• Sequencing model organism DNA

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Strategies for project

• IHGSC
• Hierarchical shotgun sequencing

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BAC assembly

• BAC clones were digested with HindIII and the
  resulting fragments gave each BAC a unique
  fingerprint
• These were used to determine clone overlaps and
  assemble BACs into contigs
• Selected minimally overlapping set of clones
• Each clone was then shotgun sequenced after being
  broken into small 2kb fragments
• Pair ends- sequence each clone from both ends

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Strategies for project

• Celera
• To practice, sequenced entire Drosophila genome
  in 1 year
• Whose DNA did they use?
• 5 people- 2 men, 3 women 1 African-American, 1
  Asian-Chinese, 1 Hispanic-Mexican, 2 Caucasians
• Whole-genome shotgun sequencing
• Sequence random bits of genome and assemble at
  the end
• Library construction
• Made 3 plasmid libraries from each donor- with
  inserts of different sizes- 2 kb, 10 kb, 50 kb
• Goal is to get 500 bp sequence data from both
  ends of each clone

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Celera genome assembly

• Used 2 approaches
• Computational combining of their data with
  sequence from GenGank- whole genome assembly
• Took their random data and combined it with the
  data from the IHGP which had been disassembled
  and used their algorithms to assemble everything
• Clustering of all fragments to regions based on
  mapping info, called compartmentalized assembly
  process
• Used STS maps to orient their contigs

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Celera method

• Screener
• Marks all microsatellites with less than 6 bp
  repeats, and screens out all known repeat
  elements (Alu, Line, rRNA)
• Overlapper
• Works with all marked regions
• Looks for end to end overlaps of at least 40 bp
  and assembles contigs
• Scaffolder
• Uses mate-pair info to link contigs together into
  scaffolds

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Whole genome shotgun sequencing
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Sequencing techniques

• Sanger dideoxy method
• Perform 4 parallel reactions, one example is
  shown here
• Dideoxy nucleotides block further chain extension

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4 colour sequencing

• Greatly improved ability to obtain large amounts
  of sequence
• Need only one lane/gel
• Use either fluor. labelled primers or chain
  terminating analogs

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Genome coverage

• Gaps in sequence
• Some easy to fix with PCR, others more difficult
• Genome size
• Estimate 3200 Mb
• Euchromatin estimate 2.9 Gb
• Total amount of finished sequence is 1 Gb

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Genome browsers

• http//genome.ucsc.edu
• http//www.ensembl.org

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Gene content of the human genome

• Only about 30,000-40,000 protein-coding genes!
• But, the genes are more complex than in lower
  organisms
• Transcriptional control
• Alternative splicing
• Average of 2.6 transcripts/gene
• 70 of alternative splices affect coding region
  of gene, rather than just changing UTR
• Alternative splicing in last exon seen 20 of
  time.

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Titling arrays to identify and define exons
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Testing whether predicted exons are expressed as
mRNA
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The proteome

• Full set of proteins encoded by genome
• More complex than invertebrates
• Mainly b/c vertebrates have arranged pre-existing
  protein motifs into more domains structures
• Also some vertebrate-specific protein domains and
  motifs
• Domain architecture
• Linear arrangement of domains within a protein
• Can be created by shuffling, adding or deleting
  domains

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Genomic landscape features

• GC content
• Found dramatic variation in CG content from 41
  genome wide average
• Regions of high CG (50-60) next to those with
  lower CG content (30-40)
• Find strong correlation between CG content and
  gene density
• Correlation with G-banding
• 98 of clones mapping to darkest G bandsare in
  regions of low GC content (37)
• 80 of clones mapping to lightest G bbands have a
  higher GC content (45)

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Genomic landscape features

• CpG islands
• Density correlates well with gene density
• Some chromosomes have many more islands, chr. 19
  has 43 per 1 Mb sequence
• Y chr. Has only 2.9 per 1 Mb
• Average is 10.5 islands per Mb

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Repeat content

• Account for at least 50 of genome
• Of this 45 of genome is from transposable
  element derived repeats

(LINE1)
(Alu)
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Segmental duplications

• Involve the transfer of 1-200 kb blocks of DNA
• Inter-chromosomal duplications
• Segments that are duplicated among non-homologous
  chromosomes
• Intra-chromosomal duplications
• Occur within a particular chromosome or arm
• Several duplicated regions mediate chromosomal
  structural rearrangements associated with genetic
  disease
• Ex. Chr. 17 three 200 kb repeats separated by 5
  Mb give rise to recombination leading to a
  syndrome

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Comparisons between genomes

• Fly and worm genomes contain large proportion of
  novel genes (30 and 50, respectively)
• Human genome has homologues of 61 of fly
  proteome, 43 of worm, 46 of yeast
• Domain architecture
• Human proteins have additional domains, compared
  to fly and worm
• Significant proportion of domain architecture
  shared between flies and humans, but some cases
  of shared domain in worms and humans not found in
  flies
• Evolutionary question - which branched off first?

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Expansions of protein families between species
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Genome and diseases

• At least 30 genes have been positionally cloned
  through using data from genome project
• Sequence has revealed some mechanisms leading to
  deletion syndromes
• Recurrent deletions due to unequal crossing over
  between duplications