Sequences and Sequencing

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Sequences and Sequencing

• Debra T. Burhans, Ph.D.
• Director, Bioinformatics Program
• Canisius College
• burhansd_at_canisius.edu
• Bioinformatics Workshop for TeachersUniversity
  at Buffalo
• August 15-19, 2005

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Outline

• Making copies cloning and PCR
• How and what we sequence
• Shotgun sequencing
• Pyro sequencing
• Summary
• Textbook reference (Mount) Chapter 2

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Making Copies cloning and PCR
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Cloning

• General strategy use a biological machine to
  do the work
• Isolate the piece of sequence you want to copy
• Insert the sequence into a molecule that can
  replicate itself
• Insert that molecule into (often) a bacterium
  that multiplies quickly
• Each new generation of bacteria contains copies
  of your DNA

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Cloning illustrated
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Cloning DNA
DNA fragments are ligated into suitable vectors.
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Cloning DNA
Recombinant vectors are propagated in bacteria.
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Cloned DNA Can be Sequenced
Cloned DNA can be grown in bacteria to produce
sufficient quantities to sequence. - this is
acceptable for sequencing one or a few clones -
far too labor intensive for sequencing large
numbers of clones
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Cloned DNA can be Amplified by the PCR
The polymerase chain reaction (PCR) is a method
for amplifying specific sequences of DNA to make
many, many copies. - in 2 hours as many as one
million copies can be made - however, the
nucleotide sequences flanking the target must be
known - this can be a problem if amplifying
DNA without first cloning it
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The Polymerase Chain Reaction
The reaction is set up in vitro and contains -
template DNA - DNA polymerase (Taq) - all four
nucleotides - primers which flank the target
sequence - other goodies - appropriate
ions - label (radioactive, fluorescent or
other) if desired
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The Polymerase Chain Reaction
The reaction placed in an automated thermal
cycler. Reactions typically have three steps -
denaturation to separate the DNA strands -
typically approximately 95oC - annealing to
permit primers to bind to target - typically
approximately 60oC - actual temperature depends
on composition of primers -polymerization to
permit the enzyme to copy the template -
typically 72oC This is repeated 30 or more
times.
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The Polymerase Chain Reaction
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PCR animation

• Animation PCR, Dolan DNA Learning Center

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How and what we sequence
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What is sequenced

• Genomic DNA contains sequences that are not
  within genes. - it also contains repeated
  sequences that confound interpretation
• RNA is expressed from genes and therefore is a
  good substrate for sequencing. - however, RNA
  is typically labile, it degrades easily
• cDNA (complementary DNA) is DNA that is copied
  from RNA in the laboratory. - it represents
  genes - it is stable
• We can get protein sequences from the cDNA

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Constructing clones for genomic DNA sequencing
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DNA Sequencing
Modern DNA sequencing is done with an automated
sequecer. Modern DNA sequencers use new
technologies -fluorescent tags for each
base -permit machine basecalling -dideoxy
chain termination chemistry -efficient and
amenable to automation -capillary
electrophoresis -permits analysis of small
samples -direct output to computer -minimizes
errors and speeds process
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Sequencing is automated
Birds-eye view of one room in the DNA sequencing
facility at the Whitehead Center for Genome
Research. The facility houses more than 150
capillary sequencers and 12 custom-made robots
that perform more than 150,000 reactions per day,
corresponding to more than 50 million raw bases
of DNA generated each day
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Sequencing a genome

• Video How to Sequence a Genome, NHGRI (948)

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Preparing cDNA for sequencing
cDNA preparation often exploits the polyA tail on
most mRNA.
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cDNA and ESTs

• The cDNA sequence represents what is expressed in
  a particular cell at a particular time
• This is useful for identifying genes and protein
  sequences
• Small fragments of cDNA that are long enough to
  uniquely identify the cDNA are called ESTs
  (expressed sequence tags)
• There are databases containing ESTs and cDNAs
• A full length cDNA fragment is the best evidence
  for the existence of a gene

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Shotgun sequencing
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Shotgun approach
DNA sequence is (now) typically obtained through
a shotgun approach -DNA is fragmented by
shooting it out of a small opening, e.g. the
end of a syringe -fragments are cloned in
suitable bacterial vectors -fragments are
sequenced using primers flanking fragment in
plasmid - sequence of fragments must be
reassembled - there are no physical maps to help
with reassembly
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Shotgun wins

• J. Craig Ventor proposed shotgun sequencing
• He thought that the reassembly would be made
  possible through the power of computation
• He formed his own company, Celera Genomics, to
  compete against the government funded effort to
  sequence the human genome
• Both efforts were completed ahead of schedule and
  at nearly the same time
• This has become the standard method for sequencing

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Pyro sequencing
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Newly announced technology

• Pyro sequencing is an incredibly fast new method
  of sequencing
• New York Times article a couple weeks ago
• Company that performs this technique
  http//www.pyrosequencing.com/
• Technology, Principle of Pyrosequencing
• Each time a new base is incorporated light is
  emitted and the base is called
• Means that at the end of the reaction the
  sequence is known

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Summary
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Sequencing present and future

• Not so long ago it took a year to sequence a few
  hundred bases, now an entire genome can be
  sequenced in a day
• The newest technologies will enable sequencing of
  genomes of individuals leading the way towards
  personalized medicine
• The ability to easily amplify a DNA sequence
  using PCR, creating millions of copies, has led
  to the use of DNA evidence in crime fighting
• The more genomes we sequence the more we learn
  about how different organisms are related
• The generation of sequence data has far outpaced
  our ability to analyze the data (at this point in
  time)