Automated Gene Sequencing

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Automated Gene Sequencing
Christophe Schlueter Mark McClelland CSC
x348 November 29, 2000
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Sanger's Method

• Most common method for determining nucleotide
  sequence
• Steps
• Amplification
• gt PCR
• gt Labeling
• gt Fragment separation (Electrophoresis)
• gt Detection / Data acquisition
• Automated methods use same basic steps, but are
• more efficient
• more accurate
• cheaper

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Amplification

• Setup
• Add two types of primers
• Add NTPs
• Add DNA polymerase
• Procedure ("Thermal cycling")
• Heat to 94C Separates double helix
• Cool to 42C Primers bind to start and end
• Heat to 72C Causes Polymerase Chain Reaction
• Repeat 30 times gt 1 billion times amplification

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Amplification Product Detection

• Number of strands in original sample usually not
  known
• Must detect amount of duplicated strands after
  each thermal cycle
• Basic procedure
• Dye is added which binds to primers
• Exposed to UV light, which makes dye glow
• Measured by electronic light detector
• Amount of light detected is proportional to
  number of strands

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Automated Amplification

• Usually only automate the most tedious
  procedures
• Thermal cycling (PCR)
• Product detection
• Example GeneAmp 5700
• Has 96 separate "reaction wells"
• Thermal cycling
• Uses a separate computer (PC) to control
  temperature
• Each cycle takes about 15 minutes
• Product detection
• Utilizes a very sensitive video camera
• Detects light from product detection dye
• Allows monitoring of all 96 wells simultaneously

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PCR

• Same as amplification process, except
• Uses didNTPs, which halt elongation randomly
• One didNTP per 200 NTPs
• Uses four separate samples (A, C, G, T)
• Usually requires only one thermal cycle
• Can use an automated amplification system, but
  with a different program

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Fragment Separation Indirect Methods

• Indirect methods separate fragments and then
  detect sequence afterwards
• Requires two steps
• 1. Separate fragments by size/weight
• 2. Detect sequence
• This makes indirect methods less efficient
• Requires fragments to be marked
• Example Gel electrophoresis

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Marking Fragments

• Radioactive labeling
• Phosphorescent dye
• Fluorescent dye
• Electrochemilimiscent dye

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Fragment Separation Direct Methods

• Direct methods analyze sequence during separation
• More efficient
• Less common
• Examples
• Mass Spectrometry
• Scanning Tunneling Microscope
• Capillary Array Electrophoresis
• Gel based
• Chip based