Automated DNA Sequencing - Automated DNA sequencing is based on the Sanger Coulson

method, with two notable differences from the standard procedure. The first difference concerns
the labelling of the products of polymerase chain reaction: automated procedures use fluorescent
labels in the place of radioactive labelling used in the standard procedure.

The fluorescent labels are usually attached to the four dideoxynucleotides used for chain
termination. In the four track system of automated DNA sequencing, each of the four
dideoxynucleotides is used in a separate reaction, and the products are run in four adjacent lanes
of the gel.

If a different fluorochrome is attached to each of the four dideoxynucleotides, all of them could
be used in the same reaction in place of preparing a separate reaction for each dideoxynucleotide.
This is called the single track system since the reaction products are run in a single gel lane or
capillary.

Generally, the DNA to be sequenced is subjected to thermal cycle sequencing to generate the
chain terminated polynucleotides required for sequencing. The reaction products are subjected to
polyacrylamide gel electrophoresis under denaturing conditions or loaded into a capillary filled
with a sequencing gel.

The bands produced in the polyacrylamide gel/capillary are identified with the help of a
fluorescence detector, which identifies the fluorescent signal emitted by each band. The
fluorochromes are excited by a laser beam and the resulting fluorescence signal is sensed by a
photovoltaic cell.

The resulting data are fed into a computer, which, in turn, converts these signals into the base
sequence of the DNA molecule. The sequence information could be printed out or stored in a
data storage device for future use; this is the second major deviation from the standard Sanger
Coulson procedure.

In the four track system, the sequence can be recognized from the raw data, but it has to be
interpreted using an appropriate computer programme in the single track system; this becomes
necessary because the shifts in mobility due to the different fluorochromes have to be
compensated for.

Automated DNA sequencers can read upto 96 DNA sequences in a 2 hours period, which is
extremely fast as compared to manual DNA sequencing.
Automated DNA sequencing has the following advantages over manual DNA sequencing:
(1) radioactivity is not used,
(2) gel processing after electrophoresis and autoradiography are not needed,
(3) the tedius manual reading of gels is not required as data are processed in a computer,
(4) the sequence data is directly fed into and stored in a computer,
(5) the separation of the same reaction products can be repeated to recheck the results in cases of
doubt since they can be stored for a long period of time, and
(6) it is extremely fast.
DNA Sequencing - Determination of nucleotide or base sequence of a
DNA molecule/fragment is known as DNA sequencing. At present, DNA
sequencing is possible for only 700-800 bp long DNA fragments. DNA
sequencing has become feasible as a result of the following important
developments:
(1) the availability of restriction endonucleases,
(2) the development of highly sensitive gel electrophoretic techniques,
which can separate DNA fragments differing by one nucleotide only,
(3) the gene cloning and PCR techniques making available large quantities
of individual DNA fragments, and
(4) the development of two reliable, relatively easy and rapid DNA
sequencing procedures, called (a) Maxam and Gilbert procedure, and (b)
the enzymatic procedure.

Maxam and Gilbert Procedure

In the first DNA sequencing technique, called Maxam and Gilbert
procedure, the DNA fragment to be sequenced is end labelled by the
addition of 32p-dATP either at the 5-ends (by the enzyme polynucleotide
kinase) or at the 3-ends (by the enzyme deoxynucleotidyl transferase) of its
two strands. The end-labelled fragment is now
(i) digested with a restriction endonuclease, which cleaves it into only two
fragments of unequal lengths. As a result, only one end of each of the two
fragments thus produced will be labelled. The two unequal fragments are
separated through gel electrophoresis and they are sequenced
separately.Alternatively, (ii) the end labelled fragment is denatured and its
two complementary strands are separated through gel electrophoresis. For
some

unknown reason, the two complementary strands of a DNA molecule

generally show different mobilities during gel electrophoresis. The samples
of complementary strands thus separated are sequenced separately. It may
be noted that each strand will be labelled at one end (either 5' or 3') only.

The single end labelled double or single stranded DNA samples thus
produced are subjected to base specific chemical cleavage so that in a
reaction mixture cleavage occurs only at one of the following four sites:
sites having G, C, G + A or C + T. Each DNA sample is partially digested
in four separate reaction mixtures (one each for specific cleavage at the
sites of G, C, G + A or C + T).

In these reaction mixtures, each DNA fragment/strand is expected to be

cleaved, on an average only once at anyone of the sites having the
particular base for which the reaction mixture is specific, each such site in
the DNA fragment/strand being equally likely to be cleaved.

The base specific cleavage of DNA fragments involves the following steps:

(1) modification of the concerned base,

(2) removal of the modified base from the DNA strand, and

(3) induction of strand break (break in the sugar phosphate back-bone) in

the position from which the modified base has been removed. Such a
cleavage generates a mixture of end-labelled DNA fragments of variable
lengths.

Digests of double stranded DNA are denatured before they are subjected to
A small primer sequence with a free 3'-OH group must be provided with the template strand for
DNA replication to proceed, since a free 3'-OH is absolutely essential for DNA polymerase I to
catalyse DNA replication.
Four different reaction mixtures are prepared for the replication of each DNA strand to be
sequenced. In one of the reaction systems, 2', 3'-dideoxycytidine triphosphate (ddCTP) is added in a
concentration about 1/100th of the normal deoxycytidine triphosphate present in the system. ddCTP
acts as a terminator of the polynucleotide chain being newly synthesized on the template strand.

Chain termination by ddCTP is achieved due to the fact that it (and the other 2'-3'-
dideoxynucleotides) does not have a free 3'-OH group as a result of which further nucleotides
cannot be added to the new chain. At the concentration used here, ddCTP would terminate the
newly synthesized polynucleotide chains at anyone of all the possible sites where cytosine is to be
incorporated in the new chain.

In one each of the three other reaction mixtures using the same DNA fragment as template, 2',3'-
dideoxythymidine triphosphate (ddTTP), 2',3'-dideoxyadenpsine triphosphate (ddATP), or 2',3'-
dideoxyguanosine triphosphate (ddGTP) is used as chain terminator to terminate the polynucleotide
chains at anyone of all the positions where T, A or G, respectively, are to be incorporated in the
new chain (each 2',3' -dideoxynucleotide is used in a separate reaction mixture).

The partially synthesized (due to chain termination) DNA chains from each of the above four
reaction mixtures are separated from the template strand by denaturation. The four single stranded
samples are now separately subjected to gel electrophoresis (in separate lanes of a gel) in order to
separate the strands according to their size.

The bands in the gels are developed onto an X-ray film through autoradiography. The fastest
moving fragment will be the smallest one, and each subsequent band will be one nucleotide longer
than the previous one.

Therefore, by comparing the bands of the four gel lanes thus obtained, nucleotide sequence of the
DNA fragment can be determined. The position of a band in the gel from a reaction mixture will
indicate the position of the base of which 2', 3’-dideoxynucleotide triphosphate was used as chain
terminator in that mixture.