DNA sequencing : It is the determination of the precise sequence of nucleotide’s in a sample of DNA.
Sanger dideoxy method: The most popular method for DNA sequencing is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded the (1980) Nobel prize in chemistry).
Procedure : The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with
a mixture of all four normal (deoxy) nucleotides in ample quantities
• dATP
• r/GTP
• c/CTP
• dT TP
a mixture of all four dideoxynucleotides, each present in limiting quantities and each labelled with a ” tag that fluoresces a different color:
• ddATP
• ddGTP
• ddc TP
• ddlTP
DNA polymerase I
Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines).
If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.
At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.