DNA Sequencing

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DNA Sequencing

• Dideoxy (Sanger) Method

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Sanger Sequencing

• Four ddNTP reactions run
• 95dNTPs, 5ddNTPs?
• Run each reaction in a gel lane

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Automated Method

• Dideoxy (Sanger)

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Capillary Electrophoresis

• Only one reaction needed
• Fluorescent labels for each ddNTP

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Southern Blot

• A technique used to locate a particular sequence
  of DNA within a complex mixture.

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Restriction Fragment Length Polymorphism

• Difference in patterns of restriction fragments
  caused by a slight difference in DNA sequence
  among individuals

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DNA Chip Technology(Microarray)
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Human Genome ProjectPublic Method

1. Identify STS (Sequence Tagged Sites) along each
   chromosome
2. Digest each chromosome with 2 restriction enzymes
   separately to create large overlapping fragments
3. Clone in BAC (Bacterial Artificial Chromosome)
4. Digest each large fragment into smaller fragments
   and clone in plasmids or viruses
5. Sequence each clone
6. Use computer programming to put together
   sequencing data in the right order using STS data

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Shotgun method

1. Shear DNA randomly by mechanical force to create
   overlapping fragments
2. Run the cut DNA on a gel
3. Cut out DNA fragments of 2 Kb in size
4. Clone into plasmids
5. Use primers specific to plasmids to sequence the
   cloned fragment 500 bps from each side
6. Using computer programming, assemble the
   sequences in the right order by using the
   overlapping regions

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