Essay About Dna Polymerase And Addition Of Further Nucleotides

Sanger Method of Dna Sequencing
Essay Preview: Sanger Method of Dna Sequencing
Report this essay
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 3OH 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 dNTPs, ddGTP, and DNA polymerase
“A” tube: all four dNTPs, ddATP, and DNA polymerase
“T” tube: all four dNTPs, ddTTP, and DNA polymerase
“C” tube: all four dNTPs, ddCTP, and DNA polymerase
All of the tubes contain a different ddNTP. As the DNA is synthesized,