Lecture 20: DNA sequencing, Microarrays, Tools for Gene Expression

Question 1

Maxam-Gilbert Sequencing

Answer 1

• Chemical

Based on chemical modification of bases and subsequent cleavage of modified nucleotide

Question 2

Sanger Sequencing or Dideoxy Sequencing

Answer 2

• Enzymatic
• Based on premature termination of DNA synthesis using dideoxynucleotides
• ​large scale
• Process
  
  1. DNA - first denatured
  2. hybridize oligonucleotide to template DNA
  3. template primer is distributed in four different tubes
  4. Add dNTPs & one ddNTP to each tube
  5. In vitro DNA synthesis produces a collection of fragments, each terminating with the dideoxynucleoside
  6. Fragments are denatured and separated on polyacrylamide gel. Bands are visualized by autoradiography ​
  7.

Question 3

formation of nucleotide chains

Answer 3

• Condensation reaction
• elimination of h20 and inorganic pyrophosphate
  *

Question 4

Autoradiography

Answer 4

• 32P isotope incorporated by
  
  • polynucleotide kinase: labels 5’ of oligonucleotide with kinase and gamma phosphate
  • incorporation of alpha-dNTP during synthesis

*

Question 5

Fluorescence DNA sequencing

Answer 5

• rxns terminated with 4 different fluorescing ddNTPs
• electrophoresis in single lane
  *

Question 6

Genome sequencing Process

Answer 6

1. Cut the DNA into overlapping fragments with endonucleases
2. Clone the fragments in plasmid or phage vectors
3. Sequence each fragment

Order the sequences into one overall sequence with computer software.

• contig: assembly of short individual DNA sequence reads
• scaffold: assembly of contigs

Question 7

Cycle Sequencing - Pyrosequencing (454 Technology)

Answer 7

• single fragment of DNA attached to bead
• PCR amplification in emulsion
• Bead inserted in well of fiber-optic slide
• Cycles of DNA synthesis with dGTP, TTP , dCTP, dATP
• detect released pyrophosphate (PPi) - leads to release of light
• adenosine 5’ phosphosulphate & sulphurylase: convert APS and PPi to ATP
• Luciferase and Luciferin: convert ATP to Light
• Peak height: corresponds to number of incorporated nucleotides
• *

Question 8

Gene Array

Answer 8

• analyze differences in gene expression
• Process

1. DNA (PCR products) spotted on glass slide
2. Cy3 (green) and Cy5 (red) probes hybridized to array
3. Fluorescence bound to array is read by laser scanner

Question 9

gene chip

Answer 9

synthesis of multiple oligonucleotides on silicon chip