Manual Sequencing

• Chemical sequencing. Requires double- or single-stranded DNA samples with one end that is radioacitvely labeled.

  Maxam-Gilbert Sequencing
• Base modifier when chain breaks at G.

  Dimethylsulfate
• Base modifier when chain breaks in G + A.

  Formic Acid
• Base modifier when chain breaks at T + C.

  Hydrazine
• Base modifier when chain breaks at C.

  Hydrazine + Salt
• Reaction when chain breaks at G.

  Methylates G
• Reaction when chain breaks at G + A.

  Protonates purines
• Reaction when chain breaks at T + C.

  Splits Pyrimidine Rings
• Reaction when chain breaks at C.

  Splits only C rings
• Time (min at 25 degrees celcius) when chain breaks at G.

  4 minutes
• Time (min at 25 degrees celcius) when chain breaks at G + A.

  5 mins
• Time (min at 25 degrees celcius) when chain breaks at T + C.

  8 mins
• Time (min at 25 degrees celcius) when chain breaks at C.

  8 mins
• Maxam-Gilbert Sequencing procedure:

  1. Templates are aliquoted to 4 tubes.
  2. Each tube is treated with a different chemical.
  3. Reduction using 10% piperidine
  4. Evaporation of piperidine
  5. Drying and resuspension in formamide
  6. Polyacrylamide gel separation
  7. Drying of gel and exposure to light
  8. Analysis of data
• Type of manual sequencing that is impractical for high-throughput sequencing for long fragments.

  Maxam-Gilbert Sequencing
• Hazardous reagent that is toxic and flammable.

  Piperidine
• Hazardous reagent that is toxic and combustible.

  Dimethylsulphate
• Hazardous reagent that is an irritant.

  Formic Acid
• Hazardous reagents that are flammable.

  Hydrazine

  Hydrazine + Salt
• Other term for Sanger Sequencing

  Dideoxy Chain Termination Sequencing
• Modification of the DNA replication process. It detects products using a radioactive or fluorescent labeled nicleotide.

  Sanger Sequencing
• Sanger Sequencing Procedure:

  1. 1:1 mixture of template and primer is placed into 4 separate reaction tubes with sequencing enzymes and buffer for polymerase activity.
  2. All dNTPs and 1/4 ddNTPs into each tube.
  3. DNA polymerase is added and reaction runs for 20 minutes.
  4. Addition of a stop buffer (EDTA, Formamide, Loading dye)
  5. Loading into denaturing polyacrylamide gel.
  6. Dry the gel and visualize the fragments.
  7. Analysis of data.
• Larger bands can be compressed at times, limiting the length of the sequence and is this harder to read.

  improvements to fuorescent dye technology and the introduction of thermal cyclers have led to automation of sequencing.
• Maxam-Gilbert Sequencing is named after?


  Allan Maxam and Walter Gilbert