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How DNA Sequence Is Determined?DNA fragments having a differenceof one nucleotide can be separated Polyacrylamide Gel Electrophoresison gel electrophoresis T A G C 32 P ATC GATC GAT 32 P ATC GATC GA 32 P ATC GATC G 32 P ATC GATC 32 P ATC GAT 32 P ATC GA G 32 P ATC G C 32 P ATC T 32 P AT 32 P A A But these bands can’t tell us If those band with the same the identity of the terminal terminal nucleotide can be nucleotides grouped, then it is possible to read the whole sequence Juang RH (2004) BCbasics
How to Obtain DNA FragmentsMaxam-Gilberts Method: Chemical Destroy → Cleavage method 32 P ATC GATC G AT32 P ATC GATC GAT 32 P ATC G ATC GAT Non-radioactive Specific Reaction to G (invisible) Destroy → CleavageSangers Method: Biosynthetic method 32P Keep on going 32 P Analogue ATC GA A,T,C ,G A TAGC TAGC TA ATC G or Producing various fragments TAGC TAGC TA STOP Terminated Template ATC G A TAGC TAGC TA Juang RH (2004) BCbasics
Sanger’s Method: How Terminated 5’ P R 1 5’ 1 P A ddNTP R 2 Terminated 3’ Normal Linking OH P 3 2- 5’ R P PO 4 A R 4 P 2 Phosphodiester bond R 5 P 3’ H H R 6 dideoxynuceotide Can not react 3’ Juang RH (2004) BCbasics