Recombinant DNA
Prof Peter Chantler
Molecular Biology of the Cell
DNA Sequencing by the Enzymatic Method
The predominate method used nowadays for DNA sequencing is an enzymatic technique, known as dideoxy sequencing or the Sanger method (to distinguish it from the chemical Maxam/Gilbert sequencing method). The DNA to be sequenced is used as a template for in vitro synthesis by DNA polymerase. Not only are all four normal deoxynucleotide triphosphates present within the reaction mixture (dATP, dTTP, dGTP, dCTP), but also present are dideoxynucleotides, one of each type (eg. ddATP) per sequencing reaction. Dideoxynucleotides are deoxynucleotides which lack hydroxyl groups at both the 2' and 3' positions, therefore cannot extend the chain by linking to another nucleotide. They therefore act as chain terminators.
Typically, in manual sequencing, four reactions are set up, one for each of the four dideoxy chain terminators to be used. In addition, either the primer, used to start the reaction, or one of the normal deoxynucleotides, is labelled; this could be through a radioactive atom or through a fluorescent tag. The dideoxynucleotide is present at a concentration about 200-fold less than its competing nucleotide. There is therefore a competition between deoxynucleotides and dideoxynucleotides (eg. in animation below for dA and ddA) for incorporation into the growing chain leading to a statistical representation of lengths of DNA which correspond to the first 200-500 residues complementary to the template. Four separate reactions are run and these are loaded and their components separated within four separate lanes of a denaturing gel by electrophoresis. Labelled bands will appear at each location where the dideoxynucleotide brought that particular elongation reactions to a halt. Thus, one can read the sequence directly eg. from the autoradiograph, from the bottom to the top.
Nowadays, the above procedure is usually automated. All reactions are performed robotically in a single tube and the reactions are terminated using fluorescent dideoxynucleotides.
Course
1 Molecular Cloning
a Choice
b Insertion
c DNA Library
d Screening
e Exp Library
2 DNA Sequencing
3 PCR
4 VNTRs
5 RAPD
Contacts
Prof Peter Chantler
RVC eMedia Unit
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