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Subculture colony producing insulin onto new agar plate Oligo(dT) primer Fish pancreas extract mRNA AAA n A-3’ 5’ AAA n A-...
 
 
 
 
 
DNA sequence analysis <ul><li>The dideoxy nucleotide technique (Sanger) </li></ul>
DNA sequence analysis
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  1. 3. Subculture colony producing insulin onto new agar plate Oligo(dT) primer Fish pancreas extract mRNA AAA n A-3’ 5’ AAA n A-3’ 5’ mRNA TTT n T-5’ poly(A) tail AAA n A-3’ 5’ TTT n T-5’ 3’ Reverse transcriptase dNTPs mRNA mRNA cDNA Oligo(dT) primer Alkali digestion of mRNA template/RNaseH TTT n T-5’ 3’ TTT n T-5’ AAA n A-3’ dsDNA with a ss hairpin susceptible to S1 nuclease cDNA DNA polymerase I dNTPs S1 nuclease TTT n T-5’ AAA n A-3’ dsDNA Clone fragments onto plasmid vector Recombinant plasmid dsDNA Plasmid Transform E. coli Screen colony producing insulin by replica plating on nitrocellulose membrane followed by staining using radio-labelled antibodies specific to insulin Bacterial colonies on agar plate Colony producing insulin Harvest insulin
  2. 9. DNA sequence analysis <ul><li>The dideoxy nucleotide technique (Sanger) </li></ul>
  3. 10. DNA sequence analysis

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