Sanger Method of Dna Sequencing

SANGER METHOD OF DNA SEQUENCING

DNA sequence determination is based on the high resolution separation of

nucleotides on polyacarylamide or an algarose gel. One method used in determining this

sequence is called the Sanger procedure. The components used are: a polyacarylamide or

an algarose gel, DNA polymerase, a primer (a short sequence), template DNA (the DNA

to be sequenced), deoxynucleotide triphosphate, and dideoxynucleitide phosphate. With

all of these, double stranded DNA is made continuously until all the triphosphates are

used up.

The deoxynucleotides, are deoxyadenine triphosphate, deoxyguanine triphosphate,

deoxycytocine triphosphate, and deoxythymine tryphosphate. They are the same as

nucleotides with the exception being that they contain hydrogen on the 3' carbon instead

of an OH. Therefore, when these deoxynucleitides are integrated into a sequence, they

prevent the addition of further nucleotides because there is no 3'OH terminal to which the

next nucleotide can be attached. This terminates the polymerixation of DNA.

Before the DNA is sequenced, it has to be denatured into single strands using heat.

A primer is then annealed to one of the template strands. The primer is constructed in

such a way that the 3' end is located next the DNA sequence of interest. Either the primer

or one of the nucleotides should be radioactively labeled so the final product can be

detected on a gel. As soon as the primer is attached to the DNA, the solution is split into

4 tubes labeled: "G", "A", "T", and "C" and then substrates are added to the respective

tubes:

"G" tube: all four dNTP's, ddGTP, and DNA polymerase

"A" tube: all four dNTP's, ddATP, and DNA polymerase

"T" tube: all four dNTP's, ddTTP, and DNA polymerase

"C" tube: all four dNTP's, ddCTP, and DNA polymerase

All of the tubes contain a different ddNTP. As the DNA is synthesized,

...