DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 34

chemical piperidine then catalyzes the breakage of DNA at one or two
predictable bases. Application of each reaction to adjacent lanes then
gives “ladders” which can be deciphered to reveal the DNA sequence.
Maxam-Gilbert sequencing, because it uses toxic chemicals, is not the
method of choice for large scale sequencing, but it is still used for DNA
footprinting where contact sites on DNA are identified.

Dideoxy or chain-termination sequencing
(also known as Sanger sequencing)

This is an enzymatic method (16) that relies on the fact that the incor-
poration of dideoxynucleotides (ddNTPs) rather than deoxynucleotides
(dNTPs) results in the termination of elongation of DNA catalyzed by
DNA polymerase. DNA polymerases works by elongation of a DNA
strand complementary to a template DNA, and requires a hydroxyl
(OH) group at the position for DNA polymerase to add the com-
plementary nucleotides. Since dideoxynucleotides lack a OH group
present in deoxynucleotides, the incorporation of a ddNTP terminates
elongation.
In dideoxy sequencing, denatured dsDNA or ssDNA is copied (se-
quenced) using a DNA polymerase. To copy DNA, a DNA polymerase
needs a primer and a supply of the 4 bases (dNTPs). A primer is a
short oligonucleotide sequence of DNA complementary to one strand
that “primes' the process (for dideoxy sequencing, vectors that have
so-called “universal priming sites” are generally used). To make the
sequence readable, 4 different reactions are used. In each of the 4
different reactions a ddNTP (ddATP, ddCTP, etc.) is added in limiting
concentrations. Included in each reaction is a radioactive nucleotide
(usually is used). The DNA polymerase initiates chain elon-
gation in a direction, starting at end of the oligonucleotide
primer that has been annealed to the DNA template. The DNA poly-
merase adds deoxynucleotides, which are selected by base-pair match-
ing, to the elongating DNA chain. DNA polymerases can incorporate
both dNTPs and ddNTPs as substrates. As a result, chain elongation
is terminated whenever a ddNTP is incorporated since they have no
hydroxyl groups. After incubation, the individual reactions containing
one of the 4 ddNTPs are run on separate lanes of an acrylamide DNA
sequencing gel and then exposed to film. The banding pattern reveals
the sequence of the DNA. Figure 2 shows an example of a dideoxy
sequencing reaction, with part of the corresponding sequences of two
independent clones of a given stretch of DNA. Notice that one of the
sequenced clones has a

substitution, otherwise known as a

mutation.