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 BUT 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”.
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. 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. After electrophoresis, the DNA sequence can be read directly
from the positions of the bands in the gel.
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Figure 1. Chain Termination DNA Sequencing
Summary:
(Step I) Chain termination sequencing involves the synthesis of new strands of DNA
complementary to a single-stranded template.
(step II) The template DNA is supplied with a mixture of all four deoxynucleotides, four
dideoxynucleotides--each labeled with a different color fluorescent tag, and DNA polymerase.
(step III) As all four deoxynucleotides are present, chain elongation proceeds until, by chance,
DNA polymerase inserts a dideoxynucleotide. The result is a new set of DNA chains all of
different lengths.
(step IV) The fragments are then separated by size using gel electrophoresis.
(Step V) As each labeled DNA fragment passes a detector at the bottom of the gel, the color is
recorded. The DNA sequence is then reconstructed from the pattern of colors representing each
nucleotide sequence.
Maxam-Gilbert Method:
In 1976-1977, Allan Maxam and Walter Gilbert developed a DNA sequencing method which is
also called as chemical cleavage method because it a based on chemical modification of DNA
and subsequent cleavage at specific bases.
Procedure:
The DNA to be sequenced must first be end labeled, at one end only. This is accomplished by
kinase treatment with 32P ATP, which transfers radioactive phosphorus from ATP to 5' end of
each strand, Dimethylsulphoxide (DMSO) is then added, and the labeled DNA samples heated to
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90 c°. This results in the breakdown of base paring and dissociation of DNA molecules into its
two component strands.
The two strands are separated from one another by gel electrophoresis, one strand is purified
from the gel and divided into four samples (G, A +G, T+C, and C), each of which is treated with
one of the cleavage reagents and modifying chemical reagents.
Gene Sequencing Technique: Gel Electrophoresis
Different methods of gene sequencing were developed in the late 1970s. These methods were
used to determine the nucleotide sequence of a particular DNA fragment. One of the common
methods is Gel Electrophoresis.
Gel electrophoresis is a technique used in molecular biology to separate charge bearing polymers
(proteins, RNA or DNA) under the influence of electric field. DNA electrophoresis is used to
separate DNA fragments primarily by size. The types of gels mostly used for DNA
electrophoresis are agarose (for relatively large DNA molecules) and polyacrylamide (for high
resolution of short DNA fragments).
In this process different sized DNA fragments starting from the same point and ending at
different points, are separated by "size fractionation” and gene sequence is noted. Different steps
of Gel Electrophoresis for gene sequences are as follows.
1. To obtain different sized DNA pieces:
DNA is cut into several pieces by using one or more restriction enzymes. As a result, different
sized fragments of DNA are obtained.
2. Preparation of Agarose gel:
Pure Agarose is in powdered form and is insoluble in water at room temperature. It dissolves in
boiling water. When it is dissolved in boiling water and allowed to cool down it polymerizes and
forms a gel.
3. Loading of gel with DNA fragments:
The DNA pieces are poured into a gel and an electrical charge is applied to the gel with the
positive charge at the bottom and the negative charge at the top. Because the DNA has a slightly
negative charge, the pieces of DNA will be attracted towards the bottom of the gel. The smaller
pieces, however, will be able to move more quickly and thus farther towards the bottom than the
larger pieces. The different-sized pieces of DNA will therefore be separated by size, with the
smaller pieces towards the bottom and the larger pieces towards the top.
4. Reading of nucleotide sequence from the gel:
Reading of nucleotide bases is facilitated using different colored florescent dyes. A detector
positioned near the bottom of the gel reads and records the color of the florescent label on each
band as it passes through a laser bean. A computer reads and records the nucleotide sequence
very quickly.
