DNA Sequencing-DNA sequencing is the process of determining the nucleic acid sequence ( the order

of nucleotides in DNA). It includes any method or technology that is used to determine the order of
the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing
methods has greatly accelerated biological and medical research and discovery. Knowledge of DNA
sequences has become indispensable and in numerous applied field such as:

• DIAGNOSTIC

• BIOTECHNOLOGY

• FORENSIC BIOLOGY AND

• BIOLOGICAL SYSTEMATICS

HISTORY

 The sequencing of DNA molecules began in the 1970s with development of the Maxam-Gilbert
method , and later the Sanger method.

 Originally developed by Frederick Sanger in 1975, most DNA sequencing that occurs in medical
and research laboratories today is performed using sequencers employing variations of the
Sanger method.

DNA STRUCTURE

In a strand of DNA, there are some simple units known as nucleotides. These nucleotides have a
‘backbone’ that consists of sugars and a phosphate group. The DNA bases can be one of four kinds and
they are attached to these sugars. These bases hold the important and unique genetic information for
body. These bases are:

 Adenine
 Thymine

 Cytosine
 Guanine
HOW DOES DNA SEQUENCING WORK?

-Into one lane or capillary of a sequencing machine goes a mixture of DNA from all four batches.
Because smaller molecules move through the gel faster, the DNA pieces come through the gel in
increasing order of size—each piece one base longer than the last.

HOW IS DNA SEQUENCING PERFORMED?

• DNA sequencing involves the process of figuring out the precise order of the four bases found
in one piece of DNA.

• The DNA is really just a template that is used to create a series of fragments.

• The fragments differ in length by one base and they are separated by size before the bases are
identified, which then effectively recreates the original DNA sequence.

• Each person has twenty-three pairs of chromosomes- one copy of the human genome.

• Because technology has limitations , we are limited in how many bases can be read at one
time.

• Therefore, we can’t just read each base from one end of a chromosome to the other. To make
it feasible, the chromosomes is cut down into smaller fragments.

METHODS FOR DNA SEQUENCING

1.BASIC METHOD

The DNA sample to be sequenced is combined in a tube with primer, DNA polymerase, and DNA
nucleotides (dATP, dTTP, dGTP, and dCTP). The four dye-labeled, chain-terminating dideoxy
nucleotides are added as well, but in much smaller amounts than the ordinary nucleotides.

The mixture is first heated to

The mixture is first heated to denature the template DNA (separate the strands), then cooled so
that the primer can bind to the single-stranded template. Once the primer has bound, the
temperature is raised again, allowing DNA polymerase to synthesize new DNA starting from the
primer. DNA polymerase will continue adding nucleotides to the chain until it happens to add a
dideoxy nucleotide instead of a normal one. At that point, no further nucleotides can be added, so
the strand will end with the dideoxy nucleotide.
 This process is repeated in a number of cycles. By the time the cycling is complete, it’s virtually
guaranteed that a dideoxy nucleotide will have been incorporated at every single position of the
target DNA in at least one reaction. That is, the tube will contain fragments of different lengths,
ending at each of the nucleotide positions in the original DNA (see figure below). The ends of the
fragments will be labeled with dyes that indicate their final nucleotide.

2.Next-generation sequencing

The name may sound like Star Trek, but that’s really what it’s called! The most recent set of DNA
sequencing technologies are collectively referred to as next-generation sequencing.

There are a variety of next-generation sequencing techniques that use different technologies.
However, most share a common set of features that distinguish them from Sanger sequencing:

Highly parallel: many sequencing reactions take place at the same time

Micro scale: reactions are tiny and many can be done at once on a chip

Fast: because reactions are done in parallel, results are ready much faster

Low-cost: sequencing a genome is cheaper than with Sanger sequencing

Shorter length: reads typically range from 505050 -700700700 nucleotides in length

PRINCIPLE

The principles of DNA Sequencing

The process of determining the order of the nucleotide bases along a DNA strand is called

sequencing. In 1977, twenty-four years after the discovery of the structure of DNA, two

separate methods for sequencing DNA were developed: the chain termination method

and the chemical degradation method. Both methods were equally popular to begin with,

but, for many reasons, the chain termination method is the method more commonly used

today. This method is based on the principle that single-stranded DNA molecules that

differ in length by just a single nucleotide can be separated from one another using

polyacrylamide gel electrophoresis, described earlier.

The DNA to be sequenced, called the template DNA, is first prepared as a single-stranded

DNA. Next, a short oligonucleotide is annealed, or joined, to the same position on each

template strand. The oligonucleotide acts as a primer for the synthesis of a new DNA
 strand that will be complimentary to the template DNA. This technique requires that four

nucleotide-specific reactions--one each for G, A, C, and T--be performed on four identical

samples of DNA. The four sequencing reactions require the addition of all the components

necessary to synthesize and label new DNA, including:

• A DNA template;

• A primer tagged with a mildly radioactive molecule or a light-emitting chemical;

• DNA polymerase--an enzyme that drives the synthesis of DNA;

• Four deoxynucleotides (G, A, C, T); and

• One dideoxynucleotide, either ddG, ddA, ddC, or ddT.

After the first deoxynucleotide is added to the growing complementary sequence, DNA

polymerase moves along the template and continues to add base after base. The strand

synthesis reaction continues until a dideoxynucleotide is added, blocking further

elongation. This is because dideoxynucleotides are missing a special group of molecules,

called a 3'-hydroxyl group, needed to form a connection with the next nucleotide. Only a

small amount of a dideoxynucleotide is added to each reaction, allowing different

reactions to proceed for various lengths of time, unti, by chance, DNA polymerase inserts

a dideoxynucleotide , terminating the reaction. Therefore, the result is a set of new chains,

all of different lengths.

To read the newly generated sequence, the four reactions are run side-by-side on a

polyacrylamide sequencing gel. The family of molecules generated in the presence of

ddATP are loaded into one lane of the gel and the other three families, generated with

ddCTP, ddGTP, and ddTTP, are loaded into three adjacent lanes. After electrophoresis, the

DNA sequence can be read directly from the positions of the bands in the gel.

IMPORTANCE

DNA sequencing is also important for biology research in general. Molecular biologists work with
systems, so knowing the gene sequence (correlates with structure) helps biologists to understand how
the protein functions in a system or pathway. ... Also, some DNA sequencing is used for gene selection
in crops and cloning.