DNA sequencing

DNA sequencing is the process by which the precise order of nucleotides in a piece of DNA can be
determined. Two different techniques were developed simultaneously- the chain termination method by F.
Sanger and A.R. Coulson in the UK, and the chemical degradation method by A. Maxam and W. Gilbert in the
USA. Both the techniques allow DNA sequences of several kb in length to be determined in the minimum of
time.

The Sanger- Coulson method
Sanger-Coulson method is a chain termination method and requires single-stranded DNA. The key principle of
the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.
The classical chain-termination method requires a single-stranded DNA template, a DNA primer, a DNA
polymerase, normal deoxynucleotidetriphosphates (dNTPs), and modified nucleotides (dideoxyNTPs) that
terminate DNA strand elongation. These ddNTPs will also be radioactively or fluorescently labelled (Figure-1)
for detection in automated sequencing machines. The DNA sample is divided into four separate sequencing
reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA
polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or
ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a
phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA
fragments of varying length.


Figure 1: DNA fragments are labelled with
a radioactive or fluorescent tag on the primer

The newly synthesized and labelled DNA fragments are heat denatured, and separated by size (with a
resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of
the four reactions run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by
autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In
the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of
different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after
incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the
different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.
Technical variations of chain-termination sequencing include tagging with nucleotides containing
radioactive phosphorus for radiolabelling, or using a primer labeled at the 5' end with a fluorescent dye
(Figure-1). Dye-primer sequencing facilitates reading in an optical system for faster and more economical
analysis and automation.

The Maxam- Gilbert method
In 19761977, Allan Maxam and Walter Gilbert developed a DNA sequencing method based on chemical
modification of DNA and subsequent cleavage at specific bases. MaxamGilbert sequencing rapidly became
more popular, since purified DNA could be directly used. The basis of Maxam-Gilbert technique is cleavage
of the existing DNA molecule using chemical reagents that act specifically at a particular nucleotide. So here a
primer is also not needed.
The double-stranded DNA fragment to be sequenced is first labelled by attaching a radioactive phosphorus
group to the 5' end of each strand. Dimethyl sulphoxide is then added and the labelled DNA sample heated to
90C. This results in breakdown of the base-pairing and dissociation of the DNA molecule into its two
component strands. The two strands are separated from one another by gel electrophoresis, which works on the
basis that one of the strands probably contains more purine nucleotides than the other and will therefore be
slightly heavier. One strand is purified from the gel and divided into four samples, each of which is treated
with one of the cleavage reagents. The modification and cleavage reactions are carried out under conditions
that result in one only one breakage per strand. Some of the cleaved fragments retain the
32
P label at their 5'
ends. After electrophoresis, using the same special conditions as for chain termination sequencing, the bands
visualized by autoradiography will represent these labelled fragments (Figure-2). The nucleotide sequence can
now be read from the autoradiograph exactly as for a chain termination experiment.


Figure 2: Diagram showing the bands obtained in a DNA sequencing gel

Reading the DNA sequence from the autoradiograph
To read the DNA sequence first the band that has moved the furthest is located. This represents the smallest
piece of DNA, the strand terminated by incorporation of the dideoxynucleotide at the first position in the
template. If the track in which the band located is A, the first nucleotide in the sequence is therefore A.
The next mobile band corresponds to a DNA molecule one nucleotide longer than the first. If the track is
noted as T, then the sequence so far is AT (Figure-3). The process is continued along the autoradiograph until
the individual bands become so bunched up that they can't be separated from one another.


Figure 3: Interpreting the autoradiograph of a chain termination sequencing experiment

How to build up a long DNA sequence
DNA sequencing by Sanger-Coulson or Maxam-Gilbert gives only about 400 nucleotides of sequence. To get
long DNA sequence, DNA sequencing experiments are performed with a lot of different fragments, all derived
from a single larger DNA molecule (Figure-4). These fragments should overlap, so the individual DNA
sequence will themselves overlap. The overlaps can be located by visualization and the master sequence
gradually built up. When DNA molecules are cleaved with two different restriction endonucleases, they
produce two different set of fragments as a result of which overlapping sequences are produced. But here one
drawback arises that the restriction sites may be inconveniently placed and individual fragments may be too
long to be completely sequenced.


Figure 4: Building up a long DNA sequence

RNA sequencing
DNA sequencing is much easier than RNA sequencing due to its greater stability. Sometimes RNA sequencing
takes place directly to determine the positions of modified nucleotides present. This is achieved by base-
specific cleavage of 5'- end- labelled RNA using Rnases that cleave 3' to a particular nucleotide. Limiting
amounts of enzyme and times of digestion are employed to generate a ladder of cleavage products which are
analyzed by PAGE.

Sequence databases
Over the years, many nucleic acid sequences have been determined by scientists all over the world, and most
scientific journals now require the prior submission of nucleic acid sequences to publish databases before they
will accept a paper for publication. The database managers share information and allow public access which
makes these databases extremely valuable resources. New sequences are being added to the databases at an
increasing rate, and special computer software is required to make good use of data. The two largest DNA
databases are EMBL in Europe and Gen Bank in the USA.

Restriction Fragment Length Polymorphism(RFLP)
Restriction fragment length polymorphism, or RFLP is a technique that exploits variations in homologous
DNA sequences. Two DNA molecules are identical or not can be known from their restriction maps. In RFLP
analysis, the DNA sample is digested by restriction enzymes and the resulting restriction fragments are
separated according to their lengths by agarose gel electrophoresis and transferred to a membrane via the
Southern blot procedure. Hybridization of the membrane to a labeled DNA probe then determines the length of
the fragments which are complementary to the probe. An RFLP occurs when the length of a detected fragment
varies between individuals. Each fragment length is considered an allele, and can be used in genetic analysis.
In addition to genetic fingerprinting, RFLP is an important tool in genome mapping, localization of genes for
genetic disorders, determination of risk for disease, and paternity testing.
Example : In the figure (Figure-5) below, it is shown that a small segment of the genome is being detected by
a DNA probe (thicker line). In allele "A", the genome is cleaved by a restriction enzyme at three nearby sites
(triangles), but only the rightmost fragment will be detected by the probe. In allele "a", restriction site 2 has be
en lost by a mutation, so the probe now detects the larger fused fragment running from sites 1 to 3.


Figure 5: A schematic diagram of RFLP by cleavage site loss

Applications
The most important application of RFLP analysis is it's use in screening programmes for human gene
mutations. This analysis is possible if the mutation responsible for the genetic disease also causes an RFLP, as
will occur with all deletion and insertion mutations, as well as those point mutations that result in loss of
restriction site. The presence of the RFLP is then a direct indication that the gene is defective and that the
individual may suffer from the resulting disease. This method has proved useful for diagnosing various types
of thalassaemia, which result from defects in the globin genes, and also in diagnosis of some types of
haemophilia.
Another useful method of genetic screening is RFLP linkage analysis. This doesn't depend on the
existence of an RFLP that is direct consequence of the genetic defect, but requires a recognizable RFLP is
present somewhere in the vicinity of the defective gene. If this RFLP is close enough then it will be inherited
with the defective gene. The presence of RFLP will therefore be indicative of the presence of the defective
gene.

Genetic fingerprinting
Genetic fingerprinting or DNA profiling is a technique which uses the individuality of DNA molecules to
distinguish between organisms, or to show the relationships between them. The DNA profiling technique was
first reported in 1984 by Sir Alec Jeffreys at the University of Leicester in England, and is now the basis of
several national DNA databases. Genetic fingerprinting is a specialized form of RFLP analysis that enables a
more general picture of genome structure to be obtained. Genetic fingerprinting examines a large number of
RFLPs at once. Here, a mixed probe is used, which is made up of several different DNA molecules targeting a
number of different parts of the genome. The targeted regions are tandem repeats, each of which consists of a
short sequence, up to about 64 bp in length, that is repeated numerous times in succession. These tandem
repeats have arrays of repeats with similar or identical core sequences are located at a number of places in the
genome, which means that a single probe, recognising the core sequence, can be used to type a number of
RFLPs at once. The number of repeat units in a single array is hypervariable.
Genetic fingerprinting is carried out in exactly the same way as standard RFLP analysis. Genomic DNA is
restricted, electrophoresed, transferred to a nitrocellulose or nylon membrane, and hybridized to the probe. The
difference is that here the autoradiograph displays a complex pattern of bands, reflecting the fact that the probe
hybridizes to arrays from numerous loci within the genome. The banding patterns obtained from two different
individuals are unlikely to be the same because of the hypervariability of each repeat array. Genetic
fingerprinting has the most widespread application in forensic science. Genetic fingerprinting is also used for
paternity testing.

References
1. Brown, T.A. (1998). Studying gene and genome structure.Gene cloning an introduction, 3rd Edn. Stanley
Thornes (Publishers) Ltd.
2. Singh, B.D.(2003). Biotechnology. Kalyani Publishers, New Delhi