25. DNA sequencing

Question 1

what is the sanger sequencing workflow?

Answer 1

• template dsDNA denatured
  – then cooled that primer can bind to single stranded template
  – once bound, DNA polymerase to synthesise new DNA starting from primer
• DNA polymerase continue adding nucleotides to the chain until it adds ddNTP instead of dNTP
  – no further nucleotides added
  – strand end with didoexyribonucleotide (termination)
• repeated, labelled ends of fragements with dyes incorperated onto ddNTPs, indicate final nucleotide
  – ddNTP incorperated into every position of targed DNA
• fragments run on capillary gel electrophoresis
  – laser excitation used to stimulate flourescent dye
  – DNA sequencers designed to discriminat all four flourescent dye wavelengths
• chromatogram of nucleotide
  – constructed and show as peaks

Question 2

what does sanger sequencing involve?

Answer 2

• DNA polymerase enzyme
• A primer
  – short piece of single stranded DNA binding to target DNA
  – acts as initiator of polymerase amplification
• four DNA nucleotides
  – dNTP’s: dATP, dTTP, dCTP, dGTP
• unique ingredient
  – dideoxynucleotides
  – chain-terminating variants of all nucleotides
  – ddATP, ddTTP, ddCTP, ddGTP (four different dyes)

Question 3

what are the advantages of sanger sequencing?

Answer 3

• higher quality sequence
• individual pies of DNA
  – bacterial plasmids
  – targeted DNA amplified in PCR

Question 4

what are the limitations of sanger sequencing?

Answer 4

• expensive
• inefficient on large scale

Question 5

what is NGS?

Answer 5

• next generation sequencing
• high-throughput sequencing
  – illumina sequencing
  – Roche 454 sequencing
  – ion torrent: proton / PGM sequencing
  – SOLiD sequencing

Question 6

what are the advantages of NGS?

Answer 6

• quicker
• cheaper
• medical diagnostics
• gene therapy
• bioprocess engineering
• microbial ecology

Question 7

what is the illumina sequencing workflow?

Answer 7

• prepare genomic DNA sample
  – randomly fragment genomic DNA
  – liagte adapter to both ends of fragments
• attache DNA to surface
  – bind single stranded fragments randomly to inside surface of flow cell channels
• bridge amplification
  – add unlabeled nucletoides and enzymes to initiate solid-phase bridge amplification
• fragment become double stranded
• denature double stranded molecules
• completion of amplification
  – on completion several million dense clusters of dbl stranded DNA are generated in each channel of flow cell
• first chemistry cycle (determine first base)
  – initiate first sequencing cycle
  – add all 4 labeled revisable terminators, primers, and DNA polymerase enzyme
• image of first chemistry cycle
  – laser excitation
  – capture image emitted floursescnece
• before initiating nect chemistry cycle
  – blocked 3’ terminus and flourophore from each incorperated base removed
• second chemistry cycle (determine second base)
  – initiate next sequencing cycle
  – add 4 labeles reversible terminatores and enzyme to flow cell
• image of second chemistry cycle
  – laser excitation
  – collect image data
• sequence read over multiple chemistry cycles
  – repeat cycles of sequencing to determine sequence of bases in given fragment

Question 8

what is the comparison of NGS sequencing platforms?**

Answer 8

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