DNA TECHNOLOGY

DNA technology has received a very wide following among geneticists. It has ushered in
a way of thinking, a different perspective in solving problem, an alternative in place of
conventional methods in the treatment of genetic disorders and improvement of crop plants and
animal breeds.
DNA technology has launched a revolution in biotechnology. Traditional biotechnology
that includes wine and cheese making using microbes and selective breeding of livestock
involves natural genetic processes, such as mutation and genetic recombination. However,
biotechnology based on the manipulation of organism’s DNA in vitro differs from earlier
practices by enabling scientists to modify genes and move them between organisms as distinct as
bacteria, plants and animals. Using DNA technology, scientists can make recombinant DNA and
then introduce it into cultured cells that replicate the DNA and may express its genes, yielding a
desired protein like insulin, growth hormone, and interferon.

Techniques in DNA technology

DNA technology refers to a wide range of techniques employed by geneticists to study,

probe, create, improve or manipulate the genetic material. There are two major categories of
DNA technology, one that makes use of recombinant DNA and another that does not use
recombinant DNA molecules.

DNA Technology Using Recombinant DNA

Recombinant DNA technology refers to the process of creating a DNA molecule by

joining together DNA or segments of DNA that normally do not occur together. Recombinant
DNA may be produced using restriction enzymes or electroporation.
Recombinant DNA technology using restriction enzymes involves the following major
steps: ( as shown in Figure 1)
 Two kinds of DNA molecules are obtained and isolated from two different cells. One is
usually a gene of interest that may have been obtained from a eukaryotic cell. The other
may be a bacterial plasmid.
 The two DNA are cut at a site with 4 to 8 nucleotides arranged in s specific sequence
using a particular restriction enzymes. A restriction enzyme is very specific in cutting at
the same sequence no matter where the DNA came from. Each DNA fragment generated,
therefore, will have sticky ends containing the same recognition sequences.
 Since the restriction fragments obtained from two different cells have the same
recognition sites, they may be joined weakly by hydrogen bonds. The result is a
recombinant DNA.
 The union may be made permanent by using DNA ligase to form a stronger
phosphodiester bond.
Recombinant DNA produced with restriction enzymes may be cloned through any one of
the following procedures.
 The recombinant DNA may be introduced into bacterial cells. The bacterial cells
are allowed to reproduce, as a result, several copies of the inserted eukaryotic

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 gene are made. The process are in which multiple copies of the desired gene are
produced is called gene cloning.
 The bacterial plasmid containing the desired gene may be used merely as a vector
to insert a gene of interest in other eukaryotic cells like yeasts. Gene cloning, in
this case, is accomplished, when the yeast cell containing the inserted gene
reproduces.

DNA Technology Without Recombinant DNA

Some techniques used in DNA technology do not warrant the use or creation of
recombinant DNA. For instance, geneticists may simply embark on a search for certain genes.
These genes may be isolated, identified, sequenced and eventually amplified.
The gene of interest may be obtained from two sources as follows:
a. directly from the DNA of a particular organism or
b. as complementary DNA (cDNA) produced from mRNA by reverse transcription.
If the gene is to be obtained directly from a particular organism, restriction enzymes may
used to cut the DNA into several pieces called restriction fragments. The fragments may be
isolated using the technique called gel electrophoresis. See Fig. 2.
Genes obtained through this method are usually difficult to manage since they often
contain intervening nucleotide sequences called introns. The second method, however, can help
overcome this problem. More manageable sizes of cDNA may be obtained by reverse
transcription because introns are usually removed during RNA processing.
The gene of interest may be difficult to identify from among several restriction fragments
obtained with restriction enzymes. Identification may be done into two ways:
1. by screening for the protein product if the gene was translated by the cells that
took up the gene or
2. by screening for the gene itself using a probe.
Once identified, the exact nucleotide sequence of the gene may be determined by DNA
sequencing. Large quantities of the gene may be generated through gene amplification .
Polymerase Chain Reaction (PCR) is a technique developed a few years ago that can amplify
genes without the use of bacterial cell. Multiple copies of the DNA are produced with the used of
DNA polymerase, primers and a supply of the four kinds of nucleotides.

Restriction Enzymes

Gene cloning and genetic engineering ( the direct manipulation of genes for practical purposes)
were made possible by the discovery of enzymes that cut DNA molecules at a limited number of
specific locations. These enzymes are called restriction enzymes. These were discovered in the
late 1960s by researchers studying bacteria. In nature, these enzymes protect the bacteria against
intruding DNA from other organisms, such as phages or other bacterial cells. They work by
cutting up the foreign DNA, a process called restriction. Most restriction enzymes are very
specific recognizing short nucleotide sequences in DNA molecules and cutting at specific points
with these sequences. The bacterial cell protects its own DNA from restriction enzymes by
adding methyl groups (--- CH3) to adenines or cytosines within the sequences recognized by the
restriction enzyme. Table 1 shows the ten commonly used restriction enzymes.

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Table 1. Ten Commonly Used Restriction Enzymes

Restriction Enzyme Sequence of Recognition Site Microbial Origin

Eco R1 5’ – G/AATTC – 3’ Escherichia coli

3’ – CTTAA/G – 5’

Taq 1 5’ – T/CGA – 3’ Thermus aquaticus YT1

3’ – AGC/T – 5’

Rsa 1 5’ – GT/AC – 3’ Rhodopseudomonas

3’ – CA/TG – 5’ sphaeroides

Sau 3A1 5’ - /GATC – 3’ Staphylococcus aureus 3A

3’ – CTAG/ - 5’

Bam H1 5’ – G/GATCC – 3’ Bacillus amyloliquefaciens H.

3’ – CCTAG/G – 5’

Hind III 5’ – A/AGCTT – 3’ Haemophilus influenzae

3’ – TTCGA/A – 5’

Kpn 1 5’ – GGTAC/C- 3’ Klebsiella pneumoniae

3’ – C/CATGG – 5’

Cla 1 5’ – AT/CGAT – 3’ Caryophanonlatum

3’ – TAGC/TA – 5 ‘

Bss HII 5’ – G/CGCGC – 3’ Bacillus stearothermophilus

3’ – CGCGC/G – 5’

Not 1 5’ – GC/GGCCGC – 3’ Nocardia otitidiscaviarum

3’ – CGCCGG/CG – 5’

(Source: Hartwell et al., 2000)

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 Figure 3 shows how restriction enzyme works in making recombinant DNA. A DNA
molecule containing a recognition sequence, or restriction site, for a particular restriction
enzyme is shown in the diagram. As shown in this example , most restriction sites are
symmetrical: The same 5’ → 3’ sequence of four to eight nucleotides (here six) is found on both
strands, thus running in opposite directions (antiparallel). Restriction enzymes cut covalent
phosphodiester bonds of both strands, often in staggered way. Because the target sequence
usually occurs (by chance) many times in a long DNA molecule, an enzyme will make many
cuts. Copies of a DNA molecules always yield the same set of restriction fragments when
exposed to that enzyme. This means that a restriction enzyme cuts a DNA molecule in
reproducible way.
The restriction fragments produced by restriction enzymes are double-stranded DNA
fragments with at least one single-stranded end, called a sticky end. This short extensions will
form hydrogen-bonded base pairs with complementary single-stranded stretches on other DNA
molecules cut with the same enzyme. The unions formed in this way are temporary, because only
a few hydrogen bonds hold the fragments together. The DNA fusions can be made permanent by
the enzyme DNA ligase, which seals the strands by catalyzing the formation of phosphodiester
bonds.

Electrophoresis
Electrophoresis is an essential technique in the analysis of nucleic acids. This is a
technique that separates nucleic acid fragments on the basis of size and electric charge in an
electric field. This technique is done by placing a sample on a porous substance, like a piece of
filter paper or a semisolid gel, which is placed in a solution that conducts electricity. If the two
molecules have approximately the same shape and mass, the one with the greatest net charge
migrates more rapidly toward the electrode of opposite polarity.
This technique uses polyacrylamide gels or agarose gel, which can be prepared with
various pore sizes, that allows the separation of two molecules polynucleotides of different
lengths but both negatively charged based on their phosphate groups. The smaller molecules
migrate at a faster rate through the gel than the larger molecules. The key separation is based on
the matrix (pores) of the gel, which restricts migration of larger molecules more than it restricts
smaller molecules. The resolving power is so great that polynucleotides that vary by even one
nucleotide in length are clearly separated. Once electrophoresis is complete, bands representing
the various sized molecules are identified either by autoradiography ( if a component of the
molecule is radioactive) or by the use of a fluorescent dye that binds to nucleic acids.

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 Figure 1

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 Figure 2

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 Figure 3

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