DNA and Sanger sequencing

Does this look familiar?

Desoxyribonucleic acid (DNA)

5’-ATGCACAACTTTTACAACTACAGCCCCCGCCCTGGGGCAAAACCTACGTGACGACAAT-3’    ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3’-TACGTGTTGAAAATGTTGATGTCGGGGGCGGGACCCCGTTTTGGATGCACTGCTGTTA-5’

• Biopolymer that codes for genetic information
• Monomers = nucleotides
• Bond types are phosphodiester bonds (3’-OH group)
• Complementary base pairing: (A=T; C≡G)
• Two antiparallel strands (running in opposite directions)

DNA replication fork

Direction of DNA

Four nucleotides

2’deoxyribose nucleoside triphosphate (dNTP)

2’,3’dideoxyribose nucleoside triphosphate (ddNTP)

Sanger sequencing

• based on modified nuclotides
• 2’,3’dideoxyribosee nucleoside triphoshate
• without 3’-OH group
• chain termination method
  
  

Sanger sequencing

Sanger sequencing chromatogram

Take-home message

Advantage of Sanger sequencing

• Devices are easy to use
• Results are obtained within couple of hours (overnight)
• Relatively cheep

Disadvantage of Sanger sequencing

• Few sequences are generated = low throughput technology
• Read length is limited to about 1000 nucleotides