sequencing is by far the most popular method and many commercial kits are available
for its use. However, there are certain occasions such as the sequencing of short
oligonucleotides where the Maxam and Gilbert method is more appropriate.
One absolute requirement for Sanger sequencing is that the DNA to be sequenced is
in a single-stranded form. Traditionally this demanded that the DNA fragment of
interest be inserted and cloned into a specialised bacteriophage vector termedM13
which is naturally single-stranded (Section 6.3.3). Although M13 is still universally
used the advent of the PCR has provided the means not only to amplify a region of any
genome or cDNA but also very quickly generate the corresponding nucleotide
sequence. This has led to an explosion in the accumulation of DNA sequence infor-
mation and has provided much impetus for gene discovery and genome mapping
(Section 6.9).
The Sanger method is simple and elegant and mimics in many ways the natural
ability of DNA polymerase to extend a growing nucleotide chain based on an existing
template. Initially the DNA to be sequenced is allowed to hybridise with an oligonu-
cleotide primer, which is complementary to a sequence adjacent to the 3^0 side of DNA
within a vector such as M13 or in an amplicon. The oligonucleotide will then act as a
primer for synthesis of a second strand of DNA, catalysed by DNA polymerase.
Since the new strand is synthesised from its 5^0 end, virtually the first DNA to be made
will be complementary to the DNA to be sequenced. One of the dNTPs that must be
provided for DNA synthesis is radioactively labelled with^32 Por^35 S, and so the newly
synthesised strand will be labelled.5.11.2 Dideoxynucleotide chain terminators
The reaction mixture is then divided into four aliquots, representing the four dNTPs,
A, C, G and T. In addition to all of the dNTPs being present in the A tube an analogue
of dATP is added (2^030 -dideoxyadenosine triphosphate (ddATP)) which is similar
to A but has no 3^0 hydroxyl group and so will terminate the growing chain since a
50 to 3^0 phosphodiester linkage cannot be formed without a 3^0 -hydroxyl group. The
situation for tube C is identical except that ddCTP is added; similarly the G and T tubes
contain ddGTP and ddTTP respectively (Fig. 5.37).
Since the incorporation of ddNTP rather than dNTP is a random event, the reaction
will produce new molecules varying widely in length, but all terminating at the same
type of base. Thus four sets of DNA sequence are generated, each terminating at a
different type of base, but all having a common 5^0 end (the primer). The four labelled
and chain-terminated samples are then denatured by heating and loaded next to each
other on a polyacrylamide gel for electrophoresis. Electrophoresis is performed at
approximately 70C in the presence of urea, to prevent renaturation of the DNA, since
even partial renaturation alters the rates of migration of DNA fragments. Very thin,
long gels are used for maximum resolution over a wide range of fragment lengths.
After electrophoresis, the positions of radioactive DNA bands on the gel are deter-
mined by autoradiography. Since every band in the track from the ddATP sample
must contain molecules which terminate at adenine, and those in the ddCTP terminate188 Molecular biology, bioinformatics and basic techniques