DNA Sequencing

A molecular technique to reveal the arrangement of nucleotides in polynucleotide chains...

First Generation

Wandering Spot Analysis (1973)

  • Developed by Maxam & Gilbert
  • Chemical Sequencing Method
  • Chemical Cleavage Method
  • Uses chemicals to degrade the sequence at particular nucleotides
    • Radioactive Phosphorus (P32)
    • Formic Acid (For depurinating Purines)
    • Dimethyl Sulfate (For methylating Guanines)
    • Hydrazine (For hydrolyzing Pyramidines)
    • Hydrazine with NaCl (For removing Cytosine)
    • Piperidine (For cleaving Phosphodiester backbone)
  • Upto 500 nucleotides
  • Extensive use of hazardous chemicals
  • Difficult to develop kits

Sequence: ATGCAGCGTTA

G AT
ATGCA
ATGCAGC
ATGCAGCGTTA
A+G
AT
ATGC
ATGCA
ATGCAGC
ATGCAGCGTT
C ATG
ATGCAG
C+T A
ATG
ATGCAG
ATGCAGCG
ATGCAGCGT
Chemical Degradation Method

Chain Termination Sequencing (1977)

  • Developed by Fredrick Sanger
  • Sanger Sequencing
  • Dideoxy Method
  • Sequence-by-Synthesis (SBS)
  • Uses ddNTPs to terminate the sequence
  • dNTPs at higher concentration than the ddNTPs
  • Approximately 1000 bp of sequence can be run in single reaction
  • Automated Capillary Electrophoresis
  • Major Provider: Applied Biosystems
  • Output format: ab1
  • Tools: BioEdit, Chromas, MEGA, etc.

Sequence: ATGCAGCGTTA

A A
ATGCA
ATGCAGCGTTA
T AT
ATGCAGCGT
ATGCAGCGTT
G ATG
ATGCAG
ATGCAGCG
C ATGC
ATGCAGC
Sanger Sequencing Method

Whole Genome Sequencing with Sanger's Method

WG with Sanger's Method