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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Genetic mapping

• Where and why genes are present inside
  chromosomes
• Simply means we need to locate genes in total
  genome
• A genetic map uses recombination, crossing over
  during meiosis, to determine how frequently two
  genes (or markers) are inherited together.
• Genes genotypes
  phenotypes

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• Gene map
• Linkage map physical
  map
• It tells you whether
  the presence of genes in chromosome
• 2 genes are close
• or distantly related
• No location

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Linkage map

• Genetic linkage is the tendency of genes that are
  located proximal to each other on a chromosome to
  be inherited together during meiosis.
• Genes whose loci are nearer to each other are
  less likely to be separated onto different
  chromatids during chromosomal crossover, and are
  therefore said to be genetically linked.
• In other words, the nearer two genes are on a
  chromosome, the lower is the chance of a swap
  occurring between them, and the more likely they
  are to be inherited together.

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Chromosome Theory of Linkage

• Morgan, along with Castle formulated the
  chromosome theory of linkage. It has the
  following postulates
• 1. Genes are found arranged in a linear manner in
  the chromosomes.
• 2. Genes which exhibit linkage are located on the
  same chromosome.
• 3. Genes generally tend to stay in parental
  combination, except in cases of crossing over.
• 4. The distance between linked genes in a
  chromosome determines the strength of linkage.
  Genes located close to each other show stronger
  linkage than that are located far from each
  other, since the former are less likely to enter
  into crossing over.

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• However crossing over does not occur between
  linked genes in every meiotic event, especially
  when the positions of the genes on the chromosome
  are very near one another.
• The frequency with which crossing over occurs
  between any two linked genes is proportional to
  the distance between the loci along the
  chromosome.

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• 1. At very small distances, crossover is very
  rare, and most gametes are parental.
• 2. As the distance between two genes increases,
  crossover frequency increases. More recombinant
  gametes, fewer parental gametes.
• 3. When genetic loci are very far apart on the
  same chromosome, crossing over nearly always
  occurs, and the
• frequency of recombinant gametes approaches 50
  percent.

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What is molecular marker?

• DNA sequence used to mark a particular location
  on a particular chromosomes.

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Genetic markers

• Modern genetic markers SNPs
• A genetic marker is a gene or DNA sequence with a
  known location on a chromosome that can be used
  to identify individuals or species.
• It can be described as a variation (which may
  arise due to mutation or alteration in the
  genomic loci) that can be observed.
• A genetic marker may be a short DNA sequence,
  such as a sequence surrounding a single base-pair
  change (single nucleotide polymorphism, SNP), or
  a long one, like

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What are they? Variable sites in the genome What
are their uses? Finding disease
genes Testing/estimating relationships Studying
population differences
Phenotype Genotype
Brown eyes BB or Bb
Blue eyes bb
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Physical mapping

• Cytogenetic mapping
• A cytogenetic map is the visual appearance of a
  chromosome when stained and examined under a
  microscope.
• Particularly important are visually distinct
  regions, called light and dark bands, which give
  each of the chromosomes a unique appearance.
• This feature allows a person's chromosomes to be
  studied in a clinical test known as a karyotype,
  which allows scientists to look for chromosomal
  alterations

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Physical Maps

• 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

• Produced by sequencing RNA which in turn
  transcript from genes
• RNA present Genes which are turned on in tissue
• Its called taq because they are not really
  complete sequence of genes, its only partially
  sequenced

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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.

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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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EST (expressed sequence tag)

• A unique stretch of DNA within a coding region of
  a gene that is useful for identifying full-length
  genes and serves as a landmark for mapping.
• An EST is a sequence tagged site (STS) derived
  from cDNA.
• An STS is a short segment of DNA which occurs but
  once in the genome and whose location and base
  sequence are known. STSs are detectable by the
  polymerase chain reaction (PCR), are helpful in
  localizing and orienting mapping and sequence
  data, and serve as landmarks in the physical map
  of the genome.

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Expressed-sequence tags (ESTs)

• are cDNA sequences that have been sequenced from
  either the 5 or 3 ends.
• They may contain all or part of a particular cDNA
  coding sequence,
• and are useful for identifying unknown genes,
  mapping their positions within a genome,
• and as a potential source for genetic material
  when a full-length cDNA is not available for a
  specific gene of interest.

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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.

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Gene Detection

• 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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Genome annotation

• The process of identifying the locations of
  genes and all of the coding regions in a genome
  and determining what those genes do.
• Once a genome is sequenced, it needs to be
  annotated to make sense of it.

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Gene Annotation

• 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