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Genomics

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Title: Genomics


1
Genomics
  • Class Molecular Biology, GIBMS 2004
  •  Source Molecular Biology by Robert F. Weaver
  • 2nd Edition, McGraw Hill Publishing, 2002

2
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

3
Sequencing of Genomes
  • 1977 Fred Sanger fX 174 bacteriophage 5,375 nt
  • Concept of ORF as coding region
  • Amino acid sequence of phage proteins
  • Overlapping genes Figure 24-1 only in viruses
  • 1995 Craig Venter Hamilton Smith
  • Haemophilus influenzae (1,830,137 nt) (1st free
    living)
  • Mycoplasma genitalium (smallest free-living,
    580,000 nt 470 genes)
  • 1996 Saccharomyces cerevisiae (1st eukaryote)
    12,068,000 nt
  • 1997 Escherichia coli 4,639,221 nt Genetically
    more important
  • Many firsts followed
  • 1999 Human chromosome 22 53,000,000 nt
  • 2000 Drosophila melanogaster 180,000,000 nt
  • 2001 Human Working draft 3,200,000,000 nt

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Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

6
Sequencing of GenomesHuman Genome Project
  • International project
  • Controversial proposed in 1990
  • Sizes and costs (500,000 pages just to print,
    time to read them?)
  • Social implications ? More so
  • Approaches
  • Systematic and conservative Francis Collins
    expected done by 2005
  • 1998 Craig Venter Celera (VitaGenomics
    Taiwan) by 2000 using shotgun sequencing ?
    needs powerful computer
  • Rough drafts of Human Genome
  • Announced June 26, 2000
  • 3,200,000,000 nt 85-99 complete

7
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

8
Sequencing of GenomesVectors for Large-Scale
Genome Project
  • Vectors needed Yeast bacterial artificial
    chromosomes
  • Cloning capacity cosmid 50Kb
  • Yeast artificial chromosomes (YAC) Fig. 24-2
  • Large capacity self replicating
  • 1,000,000 nt capacity
  • Inefficient Isolation Unstable (linear)
    Cryptic
  • Bacterial artificial chromosome (BAC) Fig.
    24-3
  • Based on F and F plasmids that conjugate
    between bacterial cells
  • Mobilize the whole host chromosome after
    insertion between cells
  • 300,000 nt capacity

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Constructed in 19Constructed in 1992 MCS
Multiple Cloning Site for cloning CmR for
selection
11
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

12
Sequencing of GenomesThe Clone-by-Clone Strategy
  • Mapping (genetically physically) the whole
    genome
  • Use overlapping clones ? Clone-by-Clone
    sequencing strategy
  • Looking for flag posts
  • Tools for mapping of genes
  • Restriction Fragment Length Polymorphisms
    (RFLPs) Fig. 24-4
  • Use to determine the position/location of a gene
    or a stretch of DNA
  • How to look for RFLPs?
  • Variable Number of Tandem Repeats (VNTRs)
  • Repeated sequences in tandem derived from
    minisatellites
  • Sequence Tagged Sites (STSs) Fig. 24-5
  • Short (60-1000 bp) sequences detectable by PCR
  • Microsatellites repeats of very short sequences
  • Highly polymorphic, thus genetic mapping is
    possible
  • Useful in physical mapping or locating specific
    sequence in the genome

13
2 individuals are polymorphic with respect to a
HindIII site (in red)
14
Primers for PCR were designed from sequences of
small areas of DNA that were already known
15
Sequencing of GenomesThe Clone-by-Clone Strategy
  • Tools for gene mapping landmarks that relate to
    gene positions
  • Construction of physical map with sequencing data
  • Mapping with STSs Fig. 24-6
  • Very laborious due to the sizes of the BACs
  • Radiation Hybrid Mapping
  • Ionizing radiation to create chromosome
    fragments
  • Form hybrid cells with hamster cells
  • Examine individually cloned cells
  • For mapping human chromosomes
  • A set of landmarks or signposts are needed and
    thus used to relate the positions of genes
  • 1998 STS-based maps constructed that included
    30,000 genes

16
After a number of positive BACs, one can begin
mapping by screening these BACs for STSs in
sequential manner
17
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

18
Sequencing of GenomesShotgun Sequencing
  • The shotgun sequencing strategy Fig. 24-7
  • Directly to sequencing without mapping
  • 1996 Craig Venter, Hamilton Smith, Leroy Hood
  • 500 nt/end x 300,000 BAC clones 300 million
    nts 10 total human genome
  • 500 nt sequenced are dispersed around every
    5,000 kb
  • Acted as sequence-tagged connector (STC) for
    each BAC clone
  • Each of the 300,000 clones connects via STC to
    30 other clones
  • Fingerprinting of each clones
  • BAC walking

19
lt1gt BAC library lt2gt Plasmid library lt3gt
Fingerprinting lt4gt BAC walking   Powerful
computer
20
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

21
Sequencing of GenomesProgress in Sequencing the
Human Genome
  • Progress Working draft 90 complete with 1
    error
  • Final draft as complete as possible with less
    than 0.01 error (1 in 10,000)
  • Functionally complete
  • 33,464,000 of the 34,491,000 nt (97.02) were
    sequenced
  • Error rate at 1 per 50,000 nt Primarily the
    22q
  • 1999 Final draft of human chromosome 22
  • 2000 Final draft of human chromosome 21
  • 2001 Working draft of whole human chromosomes
  • What do we learned from chromosome 22?
  • lt1gt still contains 11 gaps of unclonable and
    unsequenceable DNA
  • lt2gt 800 genes (679 known, related pseudogenes,
    100 predicted, 225 unknown)
  • lt3gt exons account for 3 of total length
  • lt4gt recombination rates vary along the
    chromosome Fig. 24-8
  • lt5gt local and long-range duplications
  • lt6gt large regions of 22q are conserved in
    mouse Fig. 24-9

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Sequencing of GenomesProgress in Sequencing the
Human Genome
  • 1999 Final draft of human chromosome 22
  • 2000 Final draft of human chromosome 21
  • Involved in Downs Syndrome (trisomy 21)
  • Primarily from 21q, with minors from 21p
  • A total of 33,500,000 nt were sequenced (99.7
    of total length)
  • Gaps (3) also present that no sequences are
    available
  • Relatively low gene density 225 identified
    genes (127 known, 98 predicted)
  • Total number of genes estimated in human
  • 40,000 genes (based on chromosomes 21 22)
  • 30,000 genes (working draft of whole
    chromosomes)
  • Large regions of conservation between human and
    mouse chromosomes
  • Identity of gene(s) responsible for Downs
    Syndrome still unknown
  • 2001 Working draft of whole human chromosomes

25
Sequencing of GenomesProgress in Sequencing the
Human Genome
  • 1999 Final draft of human chromosome 22
  • 2000 Final draft of human chromosome 21
  • 2001 Working draft of whole human chromosomes
  • 2.9 billion (Venter et al) to 3.2 billion
    (Collins et al) nt
  • Gaps and inaccuracies, but nevertheless,
    extremely informative
  • 25,00040,000 genes (another 12,000 possible
    genes)
  • Only 2x more than fruit flies
  • Organisms complexity not proportional to gene
    numbers
  • Expression of human genome is more complex
  • Alternative splicing? 40 of genes
  • Post-translational modifications?
  • Source of human genes importation (from
    bacteria?)
  • About 50 human genome came from transposon
    action
  • all known transposons in human are inactive now

26
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

27
Genomics and Its Applications
  • Structure genomics
  • sequencing data
  • What can we use the genomic DNA sequences for?
  • Applications
  • Study the expression of large number of genes
  • Functional Genomics
  • Finding/Identify the functions of genes,
    especially in diseases
  • Positional Cloning
  • Others

28
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

29
Genomics and Its ApplicationsTechniques in
Functional Genomics
  • Blotting analysis in the past/Miniaturized the
    blotting analysis
  • in order to study the pattern of expression of
    genes
  • DNA microarray
  • 0.25-1 nL (billionth of a liter) per
    spot Fig. 24-10
  • 5,808 DNA spots/microscope slide DNA
    microchips
  • Synthesize oligonucleotides directly on glass
    chips Fig. 24-11
  • Oligonucleotide array
  • How long must a nucleotide be to uniquely
    identify a human gene
  • in a mixture of all other human genes?
  • Hybridization analysis on DNA chip Fig.
    24-12
  • 300,000 oligonucleotides in a 0.5 X 0.5 glass
    area
  • Expressing of every and all yeast gene at the
    same time has been determined
  • Serial analysis of gene expression (SAGE) Fig.
    24-13
  • Short cDNAs (tags) are synthesized from all
    mRNAs in a cell
  • Tags are linked together in clones, sequenced to
    determine the nature (expression) of them

30
1 X 3 glass microscopic slide with 5,808 tiny
spots of DNA
31
Circle reactive groups Red photosensitive
blocking agent Blue masking agent
32
Serum-starved green (3) Serum-stimulated red
(2, 4)
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34
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

35
Genomics and Its ApplicationsPositional Cloning
  • Before genomic era
  • Positional cloning is used
  • ? to look for a gene responsible for a disease
    without knowing the function of its protein
    product
  • ? to locate a gene responsible for a disease on
    the chromosome
  • Strategies of positional cloning
  • Obtain markers closely linked to the disease
  • Scan regions between markers and possible genes
  • Search for exons with exon traps technique
  • Locate CpG islands that tend to associate
    with genes
  • Other tools
  • Human Genome Project made the scanning much
    easier

36
Genomics and Its ApplicationsPositional Cloning
  • exon traps or exon amplification
    technique Fig. 24-14
  • Look for ORFs?
  • More efficiently with exon traps technique
  • Vector contains chimeric gene under SV40
    promoter control
  • Look for exons in amplified products after
    cloning of cDNA
  • All exons or ORFs contain splice sites and thus
    survive propagation in cells
  • Locate CpG islands
  • Active human genes tend to associate with
    unmethylated CpG
  • Inactive human genes are mostly methylated CpG
  • HpaII recognizes only unmethylated CCGG
  • HpaII will only cut active genes

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Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

39
Genomics and Its ApplicationsApplications of
Functional Genomics
  • Huntingtons Disease (HD)
  • Progressive nerve disorderemotional
    disturbances adventitious movements
  • Single dominant gene with linked RFLP
    identified Fig. 24-15
  • Two (2) polymorphic sites were present in
    affected families
  • Four (4) haplotypes or haploid genotypes were
    possible Fig. 24-16
  • Which haplotype is associated with the
    Hungtingtons Disease? Fig. 24-17
  • Answer Haplotype C (those with both HindIII
    sites) is strongly
  • associated with the disease
  • However, this haplotype association varies with
    families
  • RFLP can be used as a genetic marker, just like
    a gene
  • HD gene was mapped to a region on chromosome 4
    with repeats of CAG
  • Normal individuals 11-34 CAG repeats (98 has
    less than 24 repeats)
  • Affected patients gt42 CAG repeats
  • Cystic fibrosis (CF)

40
4 haplotypes (A, B, C, D) result from the
combinations of the presence or absence of the 2
HindIII sites
41
Haplotype Site 1 Site 2 FragmentsA Absent Presen
t 17.5 3.7 1.2B Absent Absent 17.5
4.9C Present Present 15.0 3.7
1.2D Present Absent 15.0 4.9
42
lt1gt Most individuals with the C haplotype
already have the disease lt2gt No disease sufferers
lack the C haplotype
43
Genomics and Its ApplicationsApplications of
Functional Genomics
  • Huntingtons Disease (HD)
  • HD gene was located to a region near the end
    of human chromosome 4
  • Identification of HD gene
  • Number of CAG repeats of a putative gene
  • Normal ranged from 11 to 34 98 had lt24
  • Diseased all have gt42, and up to 100
  • Perspective studies using animal (mouse) model
  • Applications
  • Genetic screening of potential patients
  • Gene therapy?
  • Normal function of HD gene
    (huntingtin)
  • How the expansion of CAG repeats causes
    disease
  • extra glutamines in huntingtin protein?
  • Cystic fibrosis (CF)

44
Genomics and Its ApplicationsApplications of
Functional Genomics
  • Huntingtons Disease (HD)
  • Cystic fibrosis (CF)
  • Most common lethal genetic disease affects
    Caucasian people
  • Autosomal-recessive mutation carrier rate is
    1/20
  • Affected secretory epithelia of 1/1,600 live
    births
  • Accumulation of mucus ? infections
  • Linkage to known markers was established on 7q31
  • Positional cloning chromosome walking were
    followed Fig. 24-18
  • Unclonable region
  • Chromosomal jumping (over unclonable
    regions) Fig. 24-19
  • CF gene spans 250Kb of DNA and includes at
    least 24 exons

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Genomics and Its ApplicationsApplications of
Functional Genomics
  • Huntingtons Disease (HD)
  • Cystic fibrosis (CF)
  • Identification authentication of CF gene
  • lt1gt expressed in all tissues affected by CF
  • lt2gt gene product contains membrane-spanning
    domain
  • regulates channel of ions across the membrane
  • CFTR Cystic fibrosis transmembrane conductance
    regulator
  • lt3gt most CF patients have a 3-bp deletion in
    CFTR gene
  • a phenylalanine is missing
  • Applications Transgenic animal model
  • Applications Gene therapy CFTR protein as drug

48
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

49
Genomics and Its ApplicationsOther Applications
  • Post-genomic era
  • Single Nucleotide Polymorphisms (SNPs)
  • SNPs could link to human diseases
  • Associations with
  • polygenic traits, such as intelligence
  • responses to drugs ? pharmacogenomics
  • Vast majority of SNPs locate outside genes
  • Similarities and differences between RFLPs and
    SNPs in human
  • Testing of functions of each every genes in
    microorganisms
  • intentional and targeted mutation
  • Protein-protein interactions and activities of
    gene products
  • yeast two-hybrid system

50
Subjects To Be Covered
  • Sequencing of Genomes
  • The human genome project
  • Vectors of large scale genome projects
  • The clone-by-clone strategy
  • Shotgun sequencing
  • Progress in sequencing human genome
  • Genomics and Its Applications
  • Techniques in functional genomics
  • Positional cloning
  • Applications of functional genomics
  • Other applications
  • Bioinformatics and proteomics

51
Genomics and Its ApplicationsBioinformatics
Proteomics
  • To access, analyze and interpret sequences in
    databases
  • Bioinformatics
  • Combines biology computerized data processing
    knowledge
  • Building and manipulating biological database
  • Proteomics
  • Gene ? genome, genomics
  • Transcripts ? transcriptome, transcriptomics
  • Protein ? proteome, proteomics
  • Separation of proteins 2-D P.A.G.E
  • Analysis of proteins mass spectrometry Fig.
    24-20
  • Protein (antibody) microchips

52
Matrix-assisted laser desorption-ionization
time-of-flight (MALDI-TOF) mass spectrometry
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