DNA sequencing is the determination of the precise sequence of nucleotides 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).
The 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
• dGTP
• dCTP
• dTTP
a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a ” tag
• that fluoresces a different color:
• ddATP
• ddGTP
• ddcTP
• ddTTP
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 a different color when illuminated by a laser beam and an automatic scanner provides a next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces
printout of the sequence.
Limitation : Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.
