Genomics and Proteomics

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Genomics and Proteomics

• Wudi
• 200722040142

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content

• Positional Cloning
• Sequencing Genomes
• Applications of Genomics
• Proteomics

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Positional cloning

• Positional cloning one method for the discovery
  of the genes involved in genetic traits
• Restriction Fragment Length Polymorphisms
  (RFLP)
• Exon Traps
• CpG Islands Control regions of active human
  genes tend to be associated with unmethylated CpG
  sequences, whereas the CpGs in inactive regions
  are almost always methylated. A restriction
  enzyme ( eg. Hpa?) can cut unmethylated CpGs,
  which means that active gene regions can be
  identified

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Restriction Fragment Length Polymorphisms (RFLP)
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Exon trapping
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Identifying the Gene mutated in a Human
Disease-pinpointing the gene for Huntington
disease ( HD )

• HD is a progressive nerve disorder.
• HD disease is controlled by a single dominant
  gene.
• We do not know the nature of the product of the
  HD gene, so we could not look for the gene
  directly
• So we would like to look for a gene or other
  maker that is tightly linked to HD.

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• By making mouse-human hybrid cell lines, each
  containing only a few human chromosomes. HD was
  located to the chromosomes 4.
• The decided to focus on a 500kb region that was
  highly conserved among one-third of HD patients.
• They use an exon-trapping strategy and identity a
  handful of exon clones. Use these exons to probe
  a cDNA library to identify the DNA copy of
  interest.
• Selected gene was introduced into transgene mouse
  and investigate the phenotype.

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The RFLP associated with the Huntington disease
gene
The presents of these two Hind? sites give rise
to these four haplotype
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Southern blots of HindIII fragments from members
of two families, hybridized to the G8 probe
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The Advantage of Genomic Data

• One of the biggest anticipated payoffs of
  genomics research will be the acceleration of
  discovery of disease genes in humans.
• Positional cloning is not obsolete
• It will be important as long as we are curious
  about finding genes responsible for traits in any
  organism
• Sequenced genomes simply make positional cloning
  much easier

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Sequencing Genomes
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Vectors for Large-Scale Genome Projects

• 1. Yeast Artificial Chromosomes
• Accommodate a large insert of DNA1M
• Several serious drawbacks inefficient hard to
  isolate from yeast cells unstable tend to
  contain scrambled inserts from more than one
  site.
• 2. Bacterial Artificial Chromosomes
• Accommodate a large insert of DNA150Kb
• Circular, supercoiled BACs resist breakage

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Yeast Artificial Chromosomes
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Bacterial Artificial Chromosomes

• Cloning sites HindIII and BamHI Selection tool
  CmR the origin of replication oriS The genes
  governing partition of plasmids to daughter
  cells ParA and ParB)

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Two Strategies used in Human Genome Project

• The Clone-by-Clone Strategy
• RFLPs, VNTRs, STSs (including ESTs and
  microsatellites) as markers
• Shotgun Sequencing

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The Clone-by-Clone Strategy

• A Map-then-Sequence strategy
• The whole genome is mapped by finding markers
  regularly spaced along each chromosome.
• Sequence the clones used in the mapping and then
  place the sequences in order so they can pieced
  together.

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Variable Number of Tandem Repeats (VNTR)

• The degree of polymorphism of a RFLP is not great
  enough.
• VNTRs derive from minisatellites (Repeat
  sequences with core 12bps or more). The number of
  minisatellites repeats easily changes from one
  individual to another. So VNTRs are highly
  polymorphic, and are more useful in mapping.
• Disadvantage they tend to bunch together at the
  ends of chromosomes, leaving the interiors of the
  chromosomes relatively devoid of markers.

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Sequence-Tagged Sites (STS)

• sequences, about 60-1000bp long, that can be
  detected by PCR
• It takes much less DNA to perform PCR than to do
  a Southern blot.

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• Microsatellites (one kind of STSs)
• STSs are useful in physical mapping or locating
  specific sequences in the genome,but they are
  worthless as markers in traditional genetic
  mapping unless they are polymorphic.
• Microsatellites (random repeat sequences with
  core usually only 2-4bp long) are ideal as
  markers for both linkage and physical mapping.
  They are highly polymorphic and also widespread
  and relatively uniformly distributed in the human
  genome.
• Scientists designed PCR primers that flank the
  repeats at each locus and got these sequences as
  mapping markers.
• ESTs (expressed sequence tags)
• These are STSs generated from cDNA libraries.
• Represent genes that are expressed in the cell
  from which the mRNAs were isolated.
• Avoid most introns

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Shotgun Sequencing Strategy

• By pass the mapping stage and goes right to the
  sequencing stage
• This method relies on a computer program to find
  areas of overlap among the sequence and piece
  them together
• STC Sequence-tagged Connector

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Shotgun-sequencing method
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Sequencing standards

• Rough draft
• Working draft 90 complete, error rate of up to
  1
• Final draft error rate of less than 1/10000 have
  as few gaps as possible

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Application of Genomics

• Structural genomics Finding out the sequences of
  genomes.
• Applications of structural genomics
• 1. Functional genomics probing the pattern of
  gene expression in a given cell type at a given
  time
• 2. Positional cloning Finding genes involved in
  genetic traits, especially genetic diseases.

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Functional Genomics Techniques

• DNA Microarrays and Microchips
• Serial Analysis of Gene Expression (SAGE)
• Deletion analysis
• Location target sites for transcription factors
• Genome-wide expression analysis

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DNA microarray

• different DNAs are spotted on one chip and
  covalently attached by ultraviolet radiation to a
  thin silane layer on top of the glass

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DNA microchip

• probe is oligonucleotides synthesized right on
  the surface of a chip

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Use DNA microarray technique to examine the
effect of serum on the RNAs made by a human cell

• Fluorescent cDNA hybridized with the probes in
  the chip. cDNAs from serum stimulated cells are
  labeled red whereas cDNAs from serum starved
  cells were labeled green. Spots 24 Genes more
  active in the presence of serum. Spots 3 Genes
  more active in the absence of serum. Spot 1 Gene
  equal active in these two conditions.

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Serial Analysis of Gene Expression

• To determine which genes are expressed in a given
  tissue and the extent of that expression.
• Short tags, characteristic of particular genes,
  are generated from cDNAs and ligated together
  between linkers.
• The ligated tags then sequenced to determine
  which genes are expressed and how abundantly

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Deletion analysis

• Create mutants to genes once at a time and grow
  the whole group of mutants together under various
  conditions to see which mutants disappear most
  rapidly .
• Yeast mutants responding to the presence of
  galactose.
• Replace genes one at a time with an antibiotic
  resistance gene and each mutants flanked by
  oligomers serve as a bar code.
• All these detected mutants are probably relative
  with yeast galactose metabolism.

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Location target sites for transcription factor

• CHIP followed by DNA microarray analysis can be
  used to identify DNA-binding sites for activator
  and other proteins.
• In organisms with small genomes, such as yeast,
  all of the intergenic regions can be included in
  the microarray.
• With large genomes, that is now impractical. To
  narrow the field, CpG islands can be used. If the
  timing or conditions of an activators activity
  are known, the genes to be activated during those
  times, or under those conditions, can be used.

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Proteomics

• Transcriptome all the transcrips an organism can
  make in its lifetime
• Transcriptomics functional genomics techniques
  that measure the levels of RNAs produced from
  many genes at a time
• Proteome all the proteins that organism makes in
  its lifetime
• Proteomics studies of an organisms proteome

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Protein Separations and Analysis

• In the past time, the best separation tools 1D
  and 2D gel electrophoresis
• But now, the best separation tools MudPit
  (multidimensional protein identification
  technology)
• After been separated,proteins can be identified
• The best method for doing that involves digestion
  of the proteins one by one with proteases,then
  identifying the resulting peptides with Mass
  spectrometry
• Someday microchip with antibodies attached may
  allow analysis of proteins in complex mixtures
  without separation.

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Principle behind MALDI-TOF
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Protein interaction

• Yeast two-hybrid analysis
• Protein microarrays
• Immunoaffinity chromatography followed by MS
• Combination of phage display and computational
  method

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