Biomedical Engineering Reference
In-Depth Information
CHAPTER 7
Sequencing and mutagenesis
basic tool of gene manipulation, for it simplifies DNA
manipulations that in the past required a great
deal of ingenuity and hard work, e.g. the creation or
elimination of cleavage sites for restriction endonu-
cleases. The importance of site-directed mutagenesis
goes beyond gene structure-function relationships,
for the technique enables mutant proteins to be
generated with very specific changes in particular
amino acids (protein engineering). Such mutants
facilitate the study of the mechanisms of catalysis,
substrate specificity, stability, etc (see p. 299
et seq
.).
Introduction
DNA sequencing is a fundamental requirement
for modern gene manipulation. Knowledge of the
sequence of a DNA region may be an end in its
own right, perhaps in understanding an inherited
human disorder. More importantly, sequence infor-
mation is a prerequisite for planning any substantial
manipulation of the DNA; for example, a computer
search of the sequence for all known restriction-
endonuclease target sites will provide a complete
and precise restriction map. Similarly, mutants are
an essential prerequisite for any genetic study and
never more so than in the study of gene structure
and function relationships.
Classically, mutants are generated by treating the
test organism with chemical or physical agents that
modify DNA (mutagens). This method of mutagene-
sis has been extremely successful, as witnessed by
the growth of molecular biology, but suffers from
a number of disadvantages. First, any gene in the
organism can be mutated and the frequency with
which mutants occur in the gene of interest can be
very low. This means that selection strategies have
to be developed. Second, even when mutants with
the desired phenotype are isolated, there is no guar-
antee that the mutation has occurred in the gene of
interest. Third, prior to the development of gene
cloning and sequencing techniques, there was no
way of knowing where in the gene the mutation had
occurred and whether it arose by a single base
change, an insertion of DNA or a deletion.
As techniques in molecular biology have devel-
oped, so that the isolation and study of a single gene
is not just possible but routine, so mutagenesis has
also been refined. Instead of crudely mutagenizing
many cells or organisms and then analysing many
thousands or millions of offspring to isolate a desired
mutant, it is now possible to specifically change any
given base in a cloned DNA sequence. This technique
is known as
site-directed mutagenesis
. It has become a
Basic DNA sequencing
The first significant DNA sequence to be obtained
was that of the cohesive ends of phage-
DNA (Wu &
Taylor 1971), which are only 12 bases long. The
methodology used was derived from RNA sequenc-
ing and was not applicable to large-scale DNA
sequencing. An improved method, plus and minus
sequencing, was developed and used to sequence
the 5386 bp phage
λ
X 174 genome (Sanger
et al.
1977a). This method was superseded in 1977 by
two different methods, that of Maxam and Gilbert
(1977) and the chain-termination or dideoxy method
(Sanger
et al.
1977b). For a while the Maxam and
Gilbert (1977) method, which makes use of chemical
reagents to bring about base-specific cleavage of
DNA, was the favoured procedure. However, refine-
ments to the chain-termination method meant that by
the early 1980s it became the preferred procedure.
To date, most large sequences have been determined
using this technology, with the notable exception of
bacteriophage T7 (Dunn & Studier 1983). For this
reason, only the chain-termination method will be
described here.
The chain-terminator or dideoxy procedure for
DNA sequencing capitalizes on two properties of
DNA polymerases: (i) their ability to synthesize faith-
fully a complementary copy of a single-stranded
DNA template; and (ii) their ability to use 2
Φ
′
,3
′
-
