DNA sequencing
                                       …ACGTGACTGAGGACCGTG
                                       CGACTGAGACTGACTGGGT
                                       CTAGCTAGACTACGTTTTA
                                       TATATATATACGTCGTCGT
                                       ACTGATGACTAGATTACAG
                                       ACTGATTTAGATACCTGAC
                                       TGATTTTAAAAAAATATT…
        DNA sequencing
   Determination of nucleotide sequence
   Two similar methods:
    1. Maxam and Gilbert method
    2. Sanger method
   They depend on the production of a mixture of 
    oligonucleotides labeled either radioactively or 
    fluorescein, with one common end and differing 
    in length by a single nucleotide at the other end
   This mixture of oligonucleotides is separated by 
    high resolution electrophoresis on polyacrilamide 
    gels and the position of the bands determined
    Maxam and Gilbert Method
 The single stranded DNA fragment to be sequenced is 
  end-labeled by treatment with alkaline phosphatase to 
  remove the 5’phosphate
 It is then followed by reaction with P-labeled ATP in the 
  presence of polynucleotide kinase, which attaches P 
  labeled to the 5’terminal
 The labeled DNA fragment is then divided into four 
  aliquots, each of which is treated with a reagent which 
  modifies a specific base
  1. Aliquot A + dimethyl sulphate, which methylates guanine residue
  2. Aliquot B + formic acid, which modifies adenine and guanine residues
  3. Aliquot C + Hydrazine, which modifies thymine + cytosine residues
  4. Aliquot D + Hydrazine + 5 mol/l NaCl, which makes the reaction specific 
  for cytosine
 The four are incubated with piperidine which cleaves the 
  sugar phosphate backbone of DNA next to the residue 
  that has been modified
        Frederick Sanger 
  •Discovered DNA sequencing by chain 
    termination method
  •Nobel Prize 1 (1958)
    – Complete amino acid 
       sequence of insulin 
  •Nobel Prize 2 (1980)
    – For DNA sequencing
           Sanger Method
  DNA synthesis using deoxy- and dideoxynucleotides 
   that results in termination of synthesis at specific 
   nucleotides
  Requires a primer, DNA polymerase, a template, a 
   mixture of nucleotides, and detection system
  Incorporation of dideoxynucleotides into growing 
   strand terminates synthesis
  Synthesized strand sizes are determined for each 
   dideoxynucleotide rxn by using gel or capillary 
   electrophoresis