Genome

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Genome

• 1. Total DNA content of haploid cell
• 2. Half DNA content of diploid cell
• 3. Measured in base pairs- Kb, Mb, Gb
• 4. Homo sapiens- 3.0 x 109bp or 3.0Gb Mus
  musculus- 2.7 x 109bp or 2.7Gb
• 5. E. coli (K-12 strain)- 4.6Mb E. coli
  (O157H7 strain)- 5.5Mb, 4.1Mb common, 0.5Mb K
  islands in K-12, 1.4Mb O islands in O157H7

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Genome Sequencing

• 1. Clone by clone method
• 2. Shotgun method
• 3. Segment sequencing

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Clone by clone

• 1. Genetic and physical mapping by observing
  genetic markers in heterozygous crosses
• 2. Genomic library prepared
• 3. Chromosome segments are cloned and arranged
  into physical maps
• 4. Smaller overlapping segments are cloned
  sequenced

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Clone by clone, cont

• 5. Genomic sequence is assembled by stringing the
  segments back together
• 6.

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Shotgun

• 1. Prokaryotes
• 2. Genomic library prepared by genetic and
  physical mapping
• 3. Genome fragmented by sonication
• 4. Sequence assembled by looking for sequence
  overlaps
• 5. Done by computer using assembler software

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Segment Sequencing

• 1. Dideoxy or Sanger method
• 2. Chromatograms
• 3. Cycle sequencing

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Dideoxy Chain Termination Method

• 1. DNA polymerization
• 2. Dideoxyribonucleotide triphosphate
• 3. Technique

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DNA Polymerization

• Polymerization from C3 OH group to C5 PO4
  group
• 2. Nucleophilic attack by 3 OH on 5 PO4 of
  incoming dNTP
• 3. dAMP

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ddNTP

• ddGTP, ddTTP, ddATP, ddCTP

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Dideoxy Technique, 1

• 1. Generate specific DNA primer
• 2. Isolate DNA denature into single strand
  fragments, 5min at 65oC
• 3. Mix primer, denatured DNA, DNA polymerase,
  dNTPs of all 4 bases
• 4. One dNTP has radioactive label, dATP, usually
  35S

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Dideoxy Technique, 2

• 5. Mixture is aliquotted into 4 tubes
• 6. Appropriate ddNTP is added to each tube. 1
  of total
• 7. DNA polymerization begins, 2min at 37oC
• 8. When ddNTP incorporated into any growing
  fragment, elongation of fragment stops because 3
  OH group absent

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Dideoxy Technique, 3

• 9. Fragments vary in length, but sequence is same
  to the 3 end
• 10. DNA fragments separated by polyacrylamide gel
  electrophoresis
• 11. Gel fixed and dried to filter paper and
  exposed to X-rays
• 12. Banding pattern observed and analyzed

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Banding Pattern Analysis
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Chromatograms

• 1. Each ddNTP coupled to different color
  fluorescent dye
• 2. Radioactive labeling, manual reading
  eliminated
• 3. Discrimination higher, single gel lane or tube
• 4. Laser reads color peak information is
  recorded by computer

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Cycle Sequencing

• 1. Uses Polymerase Chain Reaction to amplify
  template
• 2. Commercial kits perform 96 reactions
  simultaneously in 96 well plate to analyze 96
  samples at once
• 3. Capillary electrophoresis uses long, flexible,
  very thin tubes and resolves miniscule amounts of
  DNA

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