DNA Sequencing

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DNA Sequencing The Human Genome Project
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Human Genome Project

• Project began in 1990 under auspices of the US
  Dept. of Energy and National Institute of Health
• 2005 was original goal of completion - first
  draft finished early, in 2001 due to
  technological advances and private competition

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Public vs. Private - Who owns the Genome?

• Francis Collins - director of the National Human
  Genome Research Institute (pictured on right).
  He coordinated a global team of scientists.
• Craig Venter, head of Celera Genomics, (pictured
  on left) a private company that also sequenced
  the genome. Venter used controversial technique
  called shotgun sequencing, in which entire
  genome is cut into small fragments then read and
  put back together with software program

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Success!

• In February 2001, 5 years ahead of schedule,
  private and public groups separately published
  DNA sequence
• The sequence revealed some surprises

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Genomes Surprises

• Instead of the 100,000 predicted number of human
  genes, both groups approximate the number closer
  to 30,000, only 3 times as many as the fruit fly.
  This means the coding region of the DNA is
  perhaps only 1-1.5 of the genome
• Only 7 percent of protein domains or functional
  sections of protein found in people were absent
  in the roundworm and fruit fly
• Human spliceosomes may do alternative splicing,
  and make different proteins from the same gene by
  splicing exons together in different combinations

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More Surprises.

• There are more segmental duplications, or copying
  of whole blocks of genes from one chromosome to
  another than expected. Choromosome 19 is the
  biggest borrower, with blocks of genes shared
  with 16 other chromosomes
• Around 230 genes seem to have been inserted in
  our sequence by viruses or bacteria
• Repetitive sequences rich in C and G tend to be
  in and around genes, while sequences rich in A
  and T are usually between genes
• Repetitive Alu sequences, around 300 bases
  long, are the most abundant sequences in the
  genome. They may modulate the activity levels of
  nearby genes

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How is DNA Sequenced?

• In 1977, Fred Sanger developed a method for DNA
  sequencing
• Automating his technique in the mid 1980s
  rapidly increased the rate at which DNA could be
  sequenced

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Dideoxynucleotides

• Sanger sequencing uses dideoxynucleotides,
  nucleotides missing oxygens in both the 2 and 3
  position
• Without an -OH on the 3 carbon of the sugar, no
  new nucleotides can be added
• ddNTPs act as chain stoppers
• This is also the chemistry behind the AIDS drug
  AZT

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Sanger Sequencing

• DNA to be sequenced is denatured, then DNA
  polymerase, regular dNTPs, and 1 labelled ddNTP
  are added to 4 tubes
• DNA is made complementary to original strand
• DNA produced is all different lengths depending
  on when ddNTP was incorporated

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Fragments are run on a gel and Identified
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Automation of Sequencing

• Sequencing technology has advanced to be able to
  sequence approximately 100,000 bases/day

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Sequencing Animation
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What Has Been Learned from Sequencing?
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Chromosome Map