INTRODUCTION TO BIOTECHNOLOGY MCB 409

DEPARTMENT OF MICROBIOLOGY
FACULTY OF SCIENCES
UNIVERSITY OF MAIDUGURI

MCB 409

INTRODUCTION TO BIOTECHNOLOGY

2 UNITS

LECTURE NOTE

DR. MUSTAFA ALHAJI ISA

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POLYMERASE CHAIN REACTION

PCR is based on ability of DNA polymerase to synthesize complementary strand to the
template strand. As DNA polymerase can add a nucleotide only onto a 3'-OH group, it
needs an artificial DNA strand (called DNA primer) of about 18 to 25 nucleotides
complementary to 3’ end of the DNA template. The primers anneal to the single-
stranded DNA template at specific temperature (depends on primer sequence) and then
DNA-Polymerase binds to this double stranded DNA produced. The again reaction
mixture is heated to 72°C (extension); a temperature optimum for DNA- polymerase
functions. This starts synthesis of the new DNA strand. Than reaction mixture is cooled
to lower temperature for short term storage, if required. This completes one cycle. After
first cycle, one DNA molecule has become two. After multiple cycle of the PCR reaction,
the specific sequence will be accumulated in billions of copies.

The PCR reaction requires the following components

 DNA template
 DNA polymerase
 Primers
 Nucleotides (dNTPs or deoxynucleotide triphosphates)

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Procedure
There are three major steps in a PCR, which are repeated for 30 or 40 cycles. This is
done on an automated cycler, which can heat and cool the tubes with the reaction
mixture in a very short time.
1. Denaturation at 94°C:
During the heating step (denaturation), the reaction mixture is heated to 94°C for 1 min,
which causes separation of DNA double stranded. Now, each strand acts as template
for synthesis of complimentary strand.
2. Annealing at 54- 65°C:
This step consist of cooling of reaction mixture after denaturation step to 54°C, which
causes hybridization (annealing) of primers to separated strand of DNA (template).
3. Extension at 72°C:
The reaction mixture is heated to 72°C which is the ideal working temperature for the
Taq polymerase. The polymerase adds nucleotide (dNTP's) complimentary to template
on 3’ –OH of primers thereby extending the new strand.

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Steps involved in PCR

GEL ELECTROPHORESIS

Gel electrophoresis is used to separate DNA fragments according to size.

The technique uses a slab of gel that has the consistency of a very firm

gelatin and is made of either agarose, a highly purified form of agar, or

polyacrylamide. A DNA sample is put into a well in the gel; there are

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generally numerous wells in a gel so that multiple samples can be analyzed

simultaneously. As a means to eventually determine the size of the various

DNA fragments in the samples, a size standard is routinely put into a well

of the same gel. This is simply a mixture of DNA fragments of known sizes

that can be used as a basis for later comparison. The gel is then subjected

to an electrical current. DNA is negatively charged, so the fragments

migrate toward the positively charged electrode. As the DNA moves

through the gel, however, not all fragments progress at the same rate. This

is because the gel acts as a sieve, impeding the long fragments while

allowing the shorter ones to pass through more quickly. Because of the

sieve-like effect of the gel, the restriction fragments are separated

according to their size. The DNA is not visible in the gel unless it is stained.

To do this, the gel containing the separated DNA fragments is immersed in

a solution containing ethidium bromide. This dye binds DNA and

fluoresces when viewed with UV light. Each fluorescent band represents

millions of molecules of a specific sized fragment of DNA. Gel

electrophoresis can also be used to separate other macromolecules,

specifically RNA and proteins, according to their size. The basic principles

are similar to that illustrated for separating DNA, but the gel compositions

differ.

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GENE THERAPY

Gene therapy is a recombinant DNA process, in which cells are taken from
the patient, altered by adding genes, and returned to the patient. A type of
genetic surgery called somatic gene therapy may be possible. Cells of a
person with a genetic disease could be removed, cultured, and transformed
with cloned DNA containing a normal copy of the defective gene. They
could be reintroduced into the individual. If these cells become established,
the expression of the normal genes may be able to cure the patient. In the
early 1990s, gene therapy of this type was used to correct a deficiency of
the enzyme adenosine deaminase (ADA). An immune deficiency disease
patient lacking the enzyme adenosine deaminase, an enzyme that destroys
toxic metabolic by-products, had been treated. Some of the patient’s
lymphocytes were removed. Lymphocytes are a type of white blood cell
that fights infection. The lymphocytes were given the adenosine deaminase
gene with the use of a modified retrovirus—which served as a vector—and
placed back into the patient’s body. Once established in the body, the cells

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with altered genes began to make the enzyme adenosine deaminase

(ADA) and alleviated the deficiency
DNA HYBRIDIZATION TECHNIQUE

In order to compare DNA from different species, scientists use a technique

called DNA hybridization. In this technique, DNA from two species is
combined and the percent similarity can be assessed.

Principle

DNA is double stranded, and individual bases bind based on Chargaff's

rules of complementary bases: adenine pairs with thymine and cytosine
pairs with guanine. When DNA is heated, the hydrogen bonds holding the
base pairs together dissolve and the DNA separates into two single strands
during a process known as DNA melting. The temperature at which the
DNA strands separate is called the melting temperature (Tm). When DNA
is cooled, complementary bases realign and bind through hydrogen
bonding.

With increased temperatures, complementary strands melt apart to form single strands
of DNA

During DNA-DNA hybridization, the DNA of each species is heated to the

melting temperature to produce single strands. Then, a strand of DNA from
each species is combined and allowed to anneal together (recombine).

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In this case, some stretches of the two strands may form base pairs while
others don't. The two molecules can be manipulated so that they form a
hybrid or separate. The conditions favoring the formation of duplex
nucleic acid are low temperature (below the Tm), high salt, and the absence
of organic solvents. The latter two conditions raise the Tm of the hybrid
duplex so that the DNA would remain more double‐stranded. On the other
hand, higher temperatures (closer to the Tm of the hybrid) lower salt, and
the presence of organic solvents would tend to push the two strands of the
DNA apart.

The percentage of strands that are either in single strand or double strand
form can be analyzed using radiolabels fluorophores or absorption of UV
light by the sample

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MONOCLONAL AND POLYCLONAL ANTIBODY

Antibody produced artificially through genetic engineering and related
techniques. Production of monoclonal antibodies was one of the most
important techniques of biotechnology to emerge during the last quarter of
the 20th century. When activated by an antigen, a circulating B cell
multiplies to form a clone of plasma cells, each secreting identical
immunoglobulin molecules. It is such immunoglobulins—derived from the
descendants of a single B cell—that are called monoclonal antibodies.
The antibody response to a natural infection or an active immunization,
however, is polyclonal. In other words, it involves many B cells, each of
which recognizes a different antigenic determinant (epitope) of the
immunizing antigen and secretes a different immunoglobulin. Thus the
blood serum of an immunized person or animal normally contains a mixture
of antibodies, all capable of combining with the same antigen but with
different epitopes that appear on the surface of the antigen. Furthermore,
even antibodies that bind to the same epitope often have different abilities
to bind to that epitope. This makes isolating an appreciable quantity of a
particular monoclonal antibody from the polyclonal mixture extremely
difficult.

HYBRIDOMA

An astonishingly high serum concentration of a single type of

immunoglobulin is associated with multiple myeloma, a type of cancer in
which a single B cell proliferates to form a tumorous clone of antibody-
secreting cells that can multiply indefinitely, like all cancer cells. Thus the
immunoglobulins made by myelomas are monoclonal, and myeloma cells

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have been propagated to produce large quantities of monoclonal

antibodies, which have been used to study the basic nature of
immunoglobulins.

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