Human Genome Project

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Human Genome Project
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Basic Strategy

• How to determine the sequence of the roughly 3
  billion base pairs of the human genome. Started
  in 1995.
• Various side projects genetic diseases,
  variations between individuals, ethnic variation,
  comparison to other species.
• Strategy
• 1. physical map relating specific DNA markers to
  the proper chromosomal position.
• 2. Overlapping set of cloned DNAs (contigs)
• 3. sequencing and assembly
• 4. finding the genes in the sequence
• 5. annotation of gene function

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Physical Maps

• A genetic map uses recombination, crossing over
  during meiosis, to determine how frequently two
  genes (or markers) are inherited together.
• A physical map determines where a given DNA
  marker is located on the DNA of the chromosome.
• Genetic and physical maps are (supposed to be)
  colinearall the genes appear in the same order
  in both maps. But, distances are quite
  different there is very little recombination in
  the centromeres, so large DNA distances are very
  short recombination distances.
• Genetic maps using microsatellite (SSR) markers
  were used to develop physical maps the
  appropriate SSR sites were expected to be found
  on the corresponding cloned DNA.

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Sequence Tagged Sites

• a sequence tagged site (STS) is a short sequence
  that is unique in the genome.
• You obtain the sequence information from cloned
  DNA, and then locate it in the genome.
• Using PCR it is then possible to determine
  whether your STS is present in any other clone or
  cell line.
• Obtaining STS sequencing the ends of large
  cloned DNAs (BACs or YACs, for example).
• Uniqueness use the cloned DNA from the STS as a
  probe on a Southern blot of genomic DNA if the
  STS is unique, only 1 band will hybridize.
• Repetitive DNA is very common in the human
  genome, and many DNA sequences are not unique.
• A good source of unique DNA is EST clones cDNA
  made from messenger RNA.
• Size a DNA sequencing run will usually give
  500-600 bp of good, reliable sequence
  information. On the other hand, consider the
  size for the genome 3 x 109 bp. Each base is
  one of 4 choices, so a 16 bp sequence will appear
  about once in 4.3 x 109 bp. In practice, 20 bp
  is about the minimum size for good PCR
  amplification, and 24 bp is about the minimum
  that will give a good BLAST hit.

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Somatic Cell Hybrids

• Human and mouse (or hamster) cultured cells can
  be fused together using polyethylene glycol.
• The resulting fused cell is a heterokaryon it
  has 2 nuclei from different species.
• If the heterokaryon undergoes mitosis, the nuclei
  fuse.
• Human chromosomes are unstable in a mixed
  nucleus, and most of them are randomly lost. The
  mouse chromosomes all stay.
• Different cell lines can be established that
  contain different combinations of human
  chromosomes
• You can identify which human chromosomes remain
  using chromosome banding techniques.
• A good way to determine which chromosome a DNA
  sequence is on. Sometimes also for gene products
  or phenotypes.

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Radiation Hybrids

• Standard somatic cell fusions contain entire
  human chromosomes. To locate a gene more
  closely, you need to use chromosome fragments.
• Start by irradiating human cells with a
  controlled dose of X-rays chromosomes break up.
  Then, fuse the cells to mouse cells. The human
  chromosome fragments get integrated into the
  mouse chromosomes.
• Create a panel of mouse/human hybrid cell lines.
  
• The current standard panels contain about 100
  cell lines.
• Each line contains about 32 of the human genome
• Average size of human genome fragment 25 kbp
• More radiation smaller fragments
• Mapping the hybrid cell lines contain random
  human chromosome fragments, but closely linked
  sites are usually in the same cell line (same
  basic principle as recombination mapping).
• Until you have located some of the markers on the
  chromosomes, radiation hybrid mapping only gives
  you information about whether any two sequences
  are close together on the chromosome.

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Contigs

• A contig is a set of partially overlapping
  clones, a contiguous set of clones. No gaps
  between them.
• Contigs allow you to build up the sequence of the
  chromosome over much larger regions than any
  single clone.
• The first reasonably complete physical map of the
  human genome involved contigs generated by YACs
  (yeast artificial chromosomes).
• Initially, you have a collection of clones with
  no information about how they are ordered on the
  chromosome.
• Contigs are built up by using PCR to identify
  unique sequences (STS or EST) on each clone, and
  then looking for overlaps between the clones.

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Sequencing Strategy

• Once a contig map of the genome was obtained, it
  was necessary to sequence each individual clone.
• Most of the actual human genome sequencing was
  done on BAC clones, which are less prone to
  rearrangement than YAC clones. BACs are about
  100-200 kbp long.
• Large clones are generally sequenced by shotgun
  sequencing The large cloned DNA is randomly
  broken up into a series of small fragments ( less
  than 1 kb). These fragments are cloned and
  sequenced. A computer program then assembles
  them based on overlaps between the sequences of
  each clone.
• To ensure that every bit has been covered, you
  need to sequence random clones until you have
  covered each spot 5-10 times on average.

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Whole Genome Shotgun Sequencing

• Why bother with creating a large scale physical
  map all that YAC and BAC cloning, radiation
  hybrids, STS comparisons, etc? Why not just
  fragment the whole genome into 1 kb pieces,
  sequence them all, and let the computer assemble
  the whole genome?
• In practice, the genome is cloned into large
  fragments first, and then each large fragment is
  broken up for shotgun sequencing. But, the large
  fragments are not ordered no physical map or set
  of contigs is created.
• Requires a lot of overlapping coverage
• Also requires good software.
• Very successful for prokaryotic genomes (10 Mbp
  or less).
• but the human genome is 300 times larger
• Big problem repeat sequence DNA, which is
  everywhere, and especially near the centromere.
  To find overlaps between clones, you need unique
  regions.
• It remains unclear whether whole genome shotgun
  sequencing will work if there is no other
  information available to provide order. It has
  not been widely adopted for eukaryotic projects
  (so far).

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Gene Detection

• the best evidence that a given DNA sequence is
  expressed is to find an EST (cDNA copy of mRNA)
  that matches it. Large numbers of EST libraries
  have been constructed and sequenced.
• The primary result of this was to determine that
  many genes have several different intron slicing
  patterns sequences are exons in some tissues but
  introns in others.
• Homology searches, using BLAST, are a good way to
  find genes. If a DNA sequence closely matches a
  sequence from another organism, it has been
  evolutionarily conserved, and that usually means
  that it is an expressed gene.
• Exon prediction exons need to be open reading
  frames (no stop codons), and they display
  patterns of nucleotide usage different from
  random DNA. Several different programs exist,
  and they give somewhat varying results.
  Hypothetical genes are genes whose existence
  has been predicted by computer but which lacks
  any experimental or cross-species data to confirm
  it.
• a conserved hypothetical gene is a sequence
  that matches other species even though there is
  no EST or other experimental evidence for its
  expression

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Gene Annotation

• Computer predictions of gene function are
  mediocre at best. Humans, especially those who
  are experts in the field, do a much better job of
  evaluating evidence and deciding what a given
  genes function is.
• There is a big problem of too much information
  not uniformly coded or maintained. The
  scientific literature contains numerous examples
  of the same gene or protein with several
  different names, and getting common definitions
  of functions is even harder.
• To counter this, the Gene Ontology Consortium
  (GO) has created a controlled vocabulary of about
  11,000 terms.
• Every gene product (protein) can be annotated
  into three general categories
• molecular function what the protein actually
  does, such as kinase activity
• biological process what cellular process the
  protein participates in, such as signal
  transduction
• cellular component where the protein is found in
  the cell, such as integral to the plasma
  membrane
• Each gene product can have multiple descriptive
  terms.
• The terms are hierarchical more specific terms
  are contained within less specific terms.
• But, a given term can have more than one parent
  and more than one child term.

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GO Example