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Genomics

Genomics. Class: Molecular Biology, GIBMS 2004  Source: “Molecular Biology” by Robert F. Weaver 2 nd Edition, McGraw Hill Publishing, 2002. Subjects To Be Covered. Sequencing of Genomes The human genome project Vectors of large scale genome projects The clone-by-clone strategy

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Genomics

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  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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Constructed in 19Constructed in 1992 MCS: Multiple Cloning Site for cloning CmR for selection

  9. 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

  10. 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

  11. 2 individuals are polymorphic with respect to a HindIII site (in red)

  12. Primers for PCR were designed from sequences of small areas of DNA that were already known

  13. 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

  14. After a number of positive BACs, one can begin mapping by screening these BACs for STSs in sequential manner

  15. 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

  16. 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”

  17. <1> BAC library <2> Plasmid library <3> Fingerprinting <4> BAC walking Powerful computer

  18. 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

  19. 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? <1> still contains 11 gaps of “unclonable” and “unsequenceable” DNA <2> 800 genes (679 known, related & pseudogenes, 100 predicted, 225 unknown) <3> exons account for 3% of total length <4> recombination rates vary along the chromosome [Fig. 24-8] <5> local and long-range duplications <6> large regions of 22q are conserved in mouse [Fig. 24-9]

  20. Sequencing of GenomesProgress in Sequencing the Human Genome 1999; Final draft of human chromosome 22 2000; Final draft of human chromosome 21 Involved in Down’s 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 Down’s Syndrome still unknown 2001; Working draft of whole human chromosomes

  21. 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 25,000–40,000 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

  22. 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

  23. 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

  24. 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

  25. 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

  26. 1” X 3” glass microscopic slide with 5,808 tiny spots of DNA

  27. Circle: reactive groups Red: photosensitive blocking agent Blue: masking agent

  28. Serum-starved: green (#3) Serum-stimulated: red (#2, #4)

  29. 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

  30. 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

  31. 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

  32. 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

  33. Genomics and Its ApplicationsApplications of Functional Genomics Huntington’s Disease (“HD”) Progressive nerve disorder:emotional 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 Hungtington’s 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: >42 “CAG” repeats Cystic fibrosis (“CF”)

  34. 4 haplotypes (A, B, C, D) result from the combinations of the presence or absence of the 2 HindIII sites

  35. Haplotype Site 1 Site 2 FragmentsA Absent Present 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

  36. <1> Most individuals with the “C” haplotype already have the disease <2> No disease sufferers lack the “C” haplotype

  37. Genomics and Its ApplicationsApplications of Functional Genomics Huntington’s 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 <24 Diseased: all have >42, 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”)

  38. Genomics and Its ApplicationsApplications of Functional Genomics Huntington’s 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

  39. Genomics and Its ApplicationsApplications of Functional Genomics Huntington’s Disease (“HD”) Cystic fibrosis (“CF”) Identification & authentication of “CF” gene <1> expressed in all tissues affected by CF <2> gene product contains membrane-spanning domain regulates channel of ions across the membrane CFTR: Cystic fibrosis transmembrane conductance regulator <3> 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

  40. 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

  41. 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

  42. 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

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