MODERN DNA SEQUENCING

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MODERN DNA SEQUENCING
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What Why?

• Sequencing means finding the order of
  nucleotides on a piece of DNA .
• Nucleotide order determines Amino acid order, and
  by extension, protein structure and function
  (proteomics)
• An alteration in a DNA sequence can lead to an
  altered or non functional protein, and hence to a
  harmful effect in a plant or animal

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What Why, Ctd.

• Understanding a particular DNA sequence can shed
  light on a genetic condition and offer hope for
  the eventual development of treatment
• DNA technology is also extended to environmental,
  agricultural and forensic applications

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DNA Sequence variation can change the
Protein produced by a particular gene
Simple point mutations such as this can
cause altered protein shape and
function. Diseases such as Sickle Cell Anaemia
and Cystic Fibrosis are caused by point mutations
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Historically there are two main methods of DNA
sequencing Maxam Gilbert, using chemical
sequencing Sanger, using dideoxynucleotides.
Modern sequencing equipment uses the principles
of the Sanger technique.
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The Sanger Technique

• Uses dideoxynucleotides (dideoxyadenine,
  dideoxyguanine, etc)
• These are molecules that resemble normal
  nucleotides but lack the normal -OH group.

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• Because they lack the -OH (which allows
  nucleotides to join a growing DNA strand),
  replication stops.

Normally, this would be where another
phosphate Is attached, but with no -OH group, a
bond can not form and replication stops
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The Sanger method requires

• Multiple copies of single stranded template DNA
• A suitable primer (a small piece of DNA that can
  pair with the template DNA to act as a starting
  point for replication)
• DNA polymerase (an enzyme that copies DNA, adding
  new nucleotides to the 3 end of the template
• A pool of normal nucleotides
• A small proportion of dideoxynucleotides labeled
  in some way ( radioactively or with fluorescent
  dyes)

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• The template DNA pieces are replicated,
  incorporating normal nucleotides, but
  occasionally and at random dideoxy (DD)
  nucleotides are taken up.
• This stops replication on that piece of DNA
• The result is a mix of DNA lengths, each ending
  with a particular labeled DDnucleotide.
• Because the different lengths travel at
  different rates during electrophoresis, their
  order can be determined.

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• Originally four separate sets of DNA, primer and
  a single different DD nucleotide were produced
  and run on a gel.
• Modern technology allows all the DNA, primers,
  etc to be mixed and the fluorescent labeled
  DDnucleotide ends of different lengths can be
  read by a laser.
• Additionally, the gel slab has been replaced by
  polymer filled capillary tubes in modern
  equipment
• This is the basis of the sequencer used at the
  Centre for Genomics and Proteomics in the School
  of Biological Sciences at the University of
  Auckland, as seen in the next slides.

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Step 1- Before submission for sequencing DNA
purity concentration is checked with the
Nanodrop
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A Nanodrop readout of known concentration to be
run as a control
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Step 2 -Samples are received and stored in the
refrigerator and a request filed
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Cost?

• Cost is dependant on a number of factors but
  typically in 2003
• Each tube of sample DNA costs 27 to run.
• An entire set of 96 tubes from one source (the
  capacity of the present equipment) costs 960.
• The methods used will readily analyze DNA
  fragments of 500-1000 bases in length, depending
  on the quality of DNA used
• Note the dye alone to run 5000 reactions costs
  61,000

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Samples arrive in Eppendorf tubes
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Step 3 - paperwork. Each request is assigned a
well in the sample tray and volumes of primers,
water, dye, etc are calculated. A typical run
has samples from a number of researchers
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Step 4- Samples are agitated then centrifuged in
an Ultracentrifuge to be sure they are in the
bottom of their Eppendorf tubes.
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Step 5 - Reagents, etc

• Each reaction requires several reagents
• Specific primers for the DNA in question
• Fluorescent Dye attached to DD nucleotides (Big
  Dye)
• Deionised water
• DNA polymerase
• Additionally, a control sample of a known DNA
  is prepared so it can run at the same time as the
  experimental DNA

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Micropipettors come in a range of sizes. They
have disposable tips that hold tiny amounts of
required reagents.
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Step 6 - Preparing the wells

• The Sample wells are loaded with DNA to be
  sequenced. Great care needs to be taken to ensure
  that each sample goes into its assigned well.
• Reagents are added (water, dye, primers) in
  required amounts
• The sample wells are spun to ensure that the
  DNA and reagents are mixed and at the bottom of
  the sample wells.

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Sample tray and micropipettor. Each tray holds 96
samples
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Step 7 - The samples are run through a cycle
sequencing process to get the fluorescent dyes
incorporated by the DNA.The DNA and reagents are
alternately heated and cooled over a2 1/2 hour
period.
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Step 8 - Sample purification to get rid of extra
dye and salts

• Unincorporated dye and salts can interfere with
  DNA analysis and need to be removed
• Samples are centrifuged, precipitated with 95
  ethanol, centrifuged again, and drained
• The process is repeated with 70 ethanol
• Dry samples are either analyzed immediately or
  stored in the dark (light degrades the
  fluorescent dyes used)
• Just before sequencing formamide is added to
  ensure that the DNA remains linear

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Entering data from the record sheet into the
Sequencer software programme
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Step 10- The sequencer is warmed up, reagents are
refreshed and the sample tray is inserted
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Inside the sequencer
Capillary tubes
Reagents
Sample tray goes here
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The Sequencer Apparatus

• Each sample tray has 96 wells (1 per sample), and
  the analyzer (3100 model) has the capacity to
  analyze 16 wells at a time
• Robotic apparatus moves the sample tray so each
  of the 16 wells is in contact with a separate
  capillary tube filled with a polymer - this
  replaces a lane on an electrophoresis gel
• Labeled DNA from that well moves up the capillary
  tube, with smaller labeled fragments moving more
  quickly than longer ones

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The Sequencer, II

• A laser reads the fluorescent label on each
  fragment as it passes up the capillary tube
• It takes 4 hours to run 16 samples. The
  robotics then move the capillaries through a
  cleaning phase and move the tray of samples so
  the next 16 samples are processed. It takes 24
  hours to process 96 samples.
• Electronic signals from the laser go to a
  sequencer programme and are converted into an
  electronic file of the code

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The Sequencer, III

• Ambiguous readings are indicated by an N
• Printouts of each record are checked for a pass
  or fail (failure may be due to degraded primer,
  insufficient DNA, etc)
• Records are stored in a computer drop box for
  electronic collection by university staff, or are
  mailed to off-campus customers

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A schematic of sequencing
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A Sequence print-out from a control sample
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What Next?

• In some instances the section of DNA analyzed may
  be all that is needed for some research project
• In other cases the DNA fragments code is matched
  to overlapping sections of other fragments. This
  eventually can result in the entire genome of an
  organism.
• Matching is done by the use of sophisticated
  software
• The National Center for Biotechnology Information
  (http//www.ncbi.nlm.nih.gov/) maintains a number
  of searchable public DNA databases
• Sequencing is starting to be done on gene
  chips, microarrays of known DNA segments- this
  area of study is evolving rapidly

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Acknowledgements
Thanks go to Kristine Boxen of the Centre for
Genomics Proteomics Craig Millar, SBS,
Auckland University
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References

• Campbell, N., Reece, J.B., 2002, Biology 6th ed.,
  Benjamin Cummings, San Francisco
• Drlica, K., 1997, Understanding DNA Gene
  Cloning, 3rd ed., John Wiley Sons, NY
• Kreuzer, H., Massey, A., 2001, Recombinant DNA
  Biotechnology, 2nd ed., ASM Press, Washington, DC
• Turner, P.C.,et.al., 1997, Instant Notes in
  Molecular Biology, Bios, Oxford
• www.ncbi.nlm.nih.gov/about/primer/genetics_molecul
  ar.html (slide 32), used by kind permission
• Photographs by L Macdonald, 2003

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The End- Or Just the Beginning?

• Compiled by
• Linda Macdonald
• For NCEA Biology A.S. 3.6
• With support from the Royal Society
• Science, Mathematics Technology Teacher
  Fellowship Scheme