DNA is the genetic
material which contains hereditary information that passes on from generation
to generation. DNA as the transforming principle, was proved in 1944 by Avery,
McLeod and McCarty. In 1953, Watson and Crick proposed 3-D, double helical
structure of DNA which was deduced from X-ray crystallography work of Rosalind
Franklin and Maurica Wilkins.
The method of deducing
order or sequence of nucleotides i.e Adenine, Guanine, Cytosine and Thymine in
DNA is known as DNA sequencing. The information we get from sequencing is used to
analyse structure and function of protein if DNA sequence is available, used in
DNA fingerprinting, in identifying disease and mutation, in comparative and
evolutionary studies. The DNA sequencing technologies have made it possible to
analyse whole genome of an organism. The turning point in the field of
molecular genetics came when genome of bacteriophage
Phi-X174 was completely sequenced in 1977. Thereafter, genomes of many
organisms have been sequenced. In 2001, the draft sequence of human genome was
First generation sequencing
Sanger sequencing or Chain termination
method of DNA sequencing was developed by Frederick Sanger and colleagues in
1977. It is based on the principle that in addition to deoxynucleotides (dNTPs ;dATP, dGTP, dCTP, dTTP)
in reaction mixture during primer extension, dideoxynucleotides (ddNTPs) lacking
3’OH group are also present. On incorporation of ddNTPs in growing
polynucleotide chain by DNA polymerase, chain termination takes place as DNA
polymerase is unable to form phosphodiester bond between ddNTP lacking 3’OH
group and 5′ phosphate group of incoming nucleotide resulting in termination at
specific position where dNTP should otherwise be added.
sequencing, DNA should be present in ample amount so as to get enough template
during sequencing reaction. This can be done by either in vivo cloning or in vitro
cloning. In in vivo cloning, DNA is
randomly fragmented followed by cloning into high copy number plasmid vector.
The vectors are then transformed into E.coli.
The bacterial colonies thus produced contain many identical copies of single
plasmid insert. While in in vitro
cloning, amplification using PCR is carried out using primers that flank the
target region. In repeated rounds of denaturation, annealing and extension, our
gene of interest gets amplified.
sequencing is performed as follows: Four separate reactions for primer
extension are used each containing single stranded DNA as template, all the
four dNTPs, DNA polymerase, primer (radioactively labelled) and one of the four
ddNTPs in each tube. The ratio of dNTPs to ddNTPs is adjusted such that
concentration of ddNTP is lower than dNTP and thus partial incorporation of
terminator occurs. Due to presence of ddNTPs, chain termination takes place at
particular nucleotide leading to generation of fragments of different length having
similarity at 5′ end and dideoxy residue at 3′ end differing by single
nucleotide only. Electrophoresis of all the four reactions is carried out on
denaturing polyacrylamide gel and banding pattern obtained is used to read the
sequence of template.