John M. S. Bartlett.pdf - Bio-Nica.info

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of simple and improved methods for enzymatic DNA. Although the chemical method
is not widely used as the enzymatic method, it has some advantages and can be very
useful in certain situations. Because it does not rely on the hybridization of a primer,
very short sequences, such as oligonucleotides, can be analyzed. It is also useful
for analyses of DNA modifications, such as methylation, and to study DNA–protein
interactions (footprinting).
3. Enzymatic DNA Sequencing
Dideoxy sequencing reactions (Sanger method) are essentially primer extension
reactions where ddNTPs are included in the mix. In the conventional dideoxy sequencing
reaction, an oligo primer is annealed to a single-stranded DNA template and
extended by DNA polymerase to synthesize a complementary copy of single-stranded
DNA in the presence of four dNTPs, one of which is 35 S-labeled. Chain growth involves
the formation of a phosphodiester bridge between the 3′-OH at the growing end of
the primer and the 5′-phosphate group of the incorporated dNTP. Thus, overall chain
growth is in the 5′→3′ direction. The reaction also contains one of four ddNTPs that
terminate elongation when incorporated into the growing DNA chain. When a ddNTP
is incorporated at the 3′ end of the growing primer chain, the elongation is terminated
selectively at A, C, G, or T owing to the missing 3′ OH group of the primer chain. The
enzymatic method is based on the ability of DNA polymerase to use both 2′ dNTPs and
2′,3′ ddNTPs as substrates. After completion of the sequencing reactions, the products
are subjected to electrophoresis on a high-resolution denaturing polyacrylamide gel
and then autoradiographed to visualize the DNA sequence.
For this form of sequencing, the purity and concentration of the template has to be
very carefully controlled, and the template has to be rendered single stranded before
the reaction will proceed. To reliably obtain good quality sequence from PCR products,
these would first have to be cloned into a suitable vector. Any clone obtained is the
product of a single molecule of PCR product, and so the likelihood of that sequence
containing misincorporated bases is relatively high. Multiple clones have therefore to
be sequenced to produce reliable data.
4. Cycle Sequencing
The introduction of thermostable DNA polymerase had the same revolutionary
impact on sequencing protocols as in other areas (3). The ability to function at higher
temperature resulted in template remaining single stranded for longer periods, overcame
many problems of secondary structure, and allowed more stringent primer annealing
conditions resulting on less background noise in sequencing data. In addition, repeated
cycles of primer annealing and extension amplifies (linearly rather than exponentially as
for PCR) even small amounts of sample DNA to generate more template. The ability to
sequence very low template concentrations means that even relatively impure material,
such as PCR products, can be used, simply by diluting out the impurities.
5. Automation
One of the major advances in sequencing technology has been the development of
automated DNA sequencers, which automate the gel electrophoresis step, detection of
band pattern, and analysis of bands. These machines are based on the enzymatic, cycle