Intro to DNA Sequencing

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Intro to DNA Sequencing
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Definition

• A technique used to determine the sequence of
  nucleotide bases in a DNA molecule or fragment

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Quick Quiz

• DNA sequencing is used to
• Determine the sequence of nucleotide bases in a
  molecular project
• Determine the order of genes in a chromosome
• Determine whether or not PCR was successful
• Determine whether or not DNA purification was
  successful

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Application Examples

• DNA sequencing is commonly used to
• Identify useful regions of DNA for other
  applications
• Identify nucleotides in a gene and the inferred
  protein sequence
• Generate data for taxonomic analysis
• Identify gene defects associated with certain
  diseases
• Analyze whole genomes

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Non-examples

• DNA sequencing is NOT commonly used for
• DNA fingerprinting
• Making monoclonal antibodies
• Conducting stem cell research

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Quick Quiz

• Which of the following is NOT an application of
  DNA sequencing?
• Generate taxonomic data to determine evolutionary
  relationships
• Mapping Genomes
• Confirming the presence of a particular gene in
  an environmental sample
• DNA fingerprinting

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Technique 1 Maxam-Gilbert (chemical cleavage)

• A pool of identical DNA fragments is labeled at
  one end and split into four tubes
• The fragments are broken preferentially at one of
  the four nucleotides, under conditions where only
  an average of about one break is made per
  fragment
• The 4 reactions are denatured and run out on an
  acrylamide sequencing gel

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http//www.cbs.dtu.dk/staff/dave/roanoke/genetics9
80211.html
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Technique 2 Sanger (dideoxy sequencing)

• The reaction is split into 4 tubes, each
  containing
• Template DNA, primer, DNA polymerase, dNTPs (A,
  T, G, C), and a low concentration of one
  dideoxynucleotide (ddA, ddT, ddG, or ddC)
• Template DNA is denatured, a primer anneals, and
  a complementary DNA strand is extended by DNA
  polymerase
• Once incorporated, a ddNTP will block further DNA
  extension
• This results in each reaction tube containing a
  mixture of DNA strands of many different lengths
• The 4 reactions are denatured and run in separate
  lanes of an acrylamide sequencing gel

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Sanger Sequencing
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Technique 3 Dye-terminator sequencing

• Combines the Sanger method (chain termination)
  with PCR
• ddNTPs are fluorescently labeled so each one has
  a unique chromaphore
• Gel is read by a sequencer, which translates the
  DNA bands into a 4-color chromatogram

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Dye-Termination Sequencing
http//oregonstate.edu/instruction/bb492/figletter
s/FigG4.html
http//www.igh.cnrs.fr/equip/sequence/DNASEQCENTER
.html
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Dye-terminator sequencing
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Quick Quiz

• In Dye-Terminator sequencing, each ddNTP is
  labeled with a unique _________ tag.
• Bioluminescent
• Radioactive
• Fluorescent
• Intercalated

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Timeline for Human Genome Project
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Whole Genome Sequencing

• Examples of those sequenced by 2007
• Human
• Sea urchin
• Chimpanzee
• Mouse
• Rat
• Honey bee
• 2 puffer fish
• 2 fruit flies
• 2 sea squirts
• 2 roundworms
• Baker's yeast
• Escherichia coli

• More recently
• Horse
• Pig
• Chicken
• Dog
• Rhesus macaque monkey
• Over 300 sequenced to date - many are bacteria

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• END

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Resources

• http//www.dnai.org/text/mediashowcase/index2.html
  ?id608
• http//www.dnalc.org/ddnalc/resources/cycseq.html

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California State Chemistry Standards

• Grade 8
• 5a. Reactant atoms and molecules interact to
  form products with different chemical properties
• 6c. Living organisms have many different kinds
  of moleculesincluding DNA
• Grades 9-12
• 2. Biological, chemical and physical properties
  of matter result from the ability of atoms to
  form bonds from electrostatic forces
• 6. Solutions are homogenous mixtures of two or
  more substances
• 8. Chemical reaction rates depend on factors
  that influence the frequency of collision of
  reactant molecules

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California State Biology Standards

• Grade 7
• 2e. DNA is the genetic material of living
  organisms and is located in the chromosomes of
  each cell
• 3a. Genetic variation and environmental factors
  are causes of evolution and diversity of
  organisms
• Grades 9-12
• 1. Fundamental life processes of plants and
  animals depend on a variety of chemical reactions
  that occur in specialized areas of the cell
• 2. Mutation and sexual reproduction lead to
  genetic variation in a population
• 4. Genes are a set of instructions encoded in
  the DNA sequence of each organism
• 5. Genetic composition of cells can be altered
  by incorporation of exogenous DNA into the cells

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California State Investigation and
ExperimentationStandards

• Grades 7
• b. Select and use appropriate tools and
  technology
• Grade 8
• a. Plan and conduct a scientific investigation
  to test a hypothesis
• Grades 9-12
• a. Select and use appropriate tools and
  technology to perform tests, collect data,
  analyze relationships, and display data
• b. Identify and communicate sources of
  unavoidable experimental error
• c. Identify possible reasons for inconsistent
  results
• d. Formulate explanations by using logic and
  evidence
• k. Recognize the cumulative nature of scientific
  evidence

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National Standards

• Grades 6-12
• Content Standard A Science as Inquiry
•  
• Content Standard C Life Science
• Content Standard E Science and Technology