DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 33

into protein, and in eukaryotic DNA is not contiguous but interrupted by
intronic sequences. DNA sequencing is far more rapid and significantly
cheaper than protein sequencing methods, and thus is the method of
choice for most sequencing projects. The sequence of a protein can
be used to predict its function by identifying potential protein interac-
tion sites, DNA interaction sites, or motifs critical to enzymatic func-
tion or consensus enzyme recognition motifs. DNA sequencing also
allows for the rapid identification of potential regulatory elements within
genes (both upstream promoter sequences as well as intronic and
enhancers) by comparing DNA sequence information with known tran-
scription factor binding sites, or evaluating non-coding sequences in the
introns around a gene that are conserved across species. These ele-
ments often predict sequences with regulatory functions. Sequencing
therefore provides basic information about protein structure that can
then be confirmed (or rejected) by directly testing these domains for
functional activities.
Two major methods have been developed for the sequencing of DNA,
one based on chemical methods (the “Maxam-Gilbert” method, named
after its developers) and an enzymatic chain termination method often
referred to as “dideoxy” sequencing or “Sanger sequencing” after its
developer. Gilbert and Sanger shared with Nobel prize for these technical
advances in 1980 with Paul Berg, who developed recombinant DNA
techniques.
Both DNA sequencing techniques rely on reactions that generate
oligonucleotide ladders, which terminate in definable nucleotides (A T
G or C), although the methods used to achieve these are quite differ-
ent. In both methods, a piece of DNA that can be manipulated in the
sequencing reactions (often generated using restriction enzymes) is in-
serted into a cloning vector. Cloning vectors contain unique sites that
flank both sides the cloned DNA fragment, the insert, to be sequenced.
These sites are complementary to oligonucleotide primers that initiate
the reactions. DNA sequencing generally relies on the use of high reso-
lution denaturing polyacrylamide gels which can resolve single stranded
DNA ladders over several hundred base pairs.
Maxam-Gilbert sequencing
This method (15) relies on the use of chemical reagents that modify
specific bases, followed by the cleavage of the DNA at one or two spe-
cific nucleotides. Labeled DNA (usually radioactively labeled at the
or ends of the strands) is subjected to one of 3 chemical reactions
which modify bases in a specific way. Depending on the modification, the