⇒ Nucleic Acid Manipulations
⇒ Nucleic Acid Hybridization
⇒ The Polymerase Chain Reaction
⇒ Nucleic Acid Sequencing
Nucleic acid sequencing reveals the genetic code of a DNA molecule. It
may be carried out using one of two methods, each of which results in the
production of DNA fragments of various lengths, differing from each other
by a single base and from which one can infer the nucleic acid sequence
of the molecule. This is accomplished using denaturing polyacrylamide
gels. Whereas agarose gels can separate DNA molecules differing in
length by 30-50 bases, polyacrylamide gels can discriminate among
DNA molecules differing in length by a single base. Denaturing gels
cause the DNA molecule to become single stranded and remain that way
throughout the entire process of electrophoresis. Denaturing gels contain
urea and are run at elevated temperatures, both of which promote the separation
of the two strands of the DNA molecule.
Again, the DNA must be labeled in order to be visualized. The most
common form of labeling is with radioactive isotopes, in particular, 32P,
33P, or 35S. After electrophoresis, the gel is dried and placed next to a
sheet of x-ray film in a dark place. During this time the radioactive particles
emitted from the isotope in each DNA molecule "expose" the film,
and after development, a dark band is seen on the film at the position
where the DNA band was located in the gel. This picture, called an autoradiograph, is a mirror image of the position of the DNA bands in the
There are two methods that can be used to sequence DNA molecules.
The Maxam-Gilbert method is based on cleavage of DNA at specific
sites by chemicals rather than enzymes. However, this method is seldom
used anymore; the Sanger method is preferred.
In the Sanger method, the enzymatic synthesis of DNA takes place
by the sequential formation of a phosphodiester bond between the free 5'
phosphate group of an incoming nucleotide and the 3' OH group of the
growing chain. This process takes place throughout the length of the
DNA molecule. Dideoxynucleotides lack a 3' OH group, and have a 3'
H group instead. In the presence of a dideoxynucleotide, the synthesis of
DNA stalls because the diphosphate bond cannot be formed. The chain
growth terminates at that point, and the last base on the 3' end of the chain
is a dideoxy terminator. This modification of Sanger's method of DNA
sequencing is known as dideoxy termination sequencing.
In the Sanger sequencing technique, four different reaction mixtures
are used to sequence a DNA fragment. Each reaction mixture contains the
template DNA molecule to be sequenced, radioactively labeled primers,
all four deoxynucleotides, DNA polymerase, and a different dideoxy terminator
(ddATP, ddCTP, ddGTP, or ddTTP). When one of these terminators
is incorporated in the newly synthesized DNA strand, it will stop
further synthesis of that strand; the result is that all the strands of various
lengths in the reaction mixture end in the same base. The radioactive
products are separated by electrophoresis and visualized by autoradiography.
Reading from the bottom of the gel (shortest fragments terminated
closest to the 5' end) upward reveals the base sequence complementary
to that of the template strand.