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Applied molecular technique
Introduction
Molecular biology is an area of biology concerned with the process of gene
transcription to yield RNA, the translation of RNA into proteins and the role those proteins
play in cellular function. Since around 1960, molecular biologists have developed methods
to identify, isolate, and manipulate molecular components in cells including DNA, RNA,
and proteins. Molecular biology deals with nucleic acids, which come in two forms:
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The chemical differences
between the two substances are minimal. They are both polymers that are made of four
building blocks each, deoxynucleotides in DNA and nucleotides in RNA. The nucleotides
of RNA are composed of a base constituent (adenine, cytosine, guanine, or uracil), a
glucose component (ribose), and a phosphoryl residue, whereby two nucleotides are
connected to each other through phosphoglucose bonds. In this way, one nucleotide can be
connected to the next, forming a long chain known as a polynucleotide, which is
designated as RNA. DNA is formed in almost the same manner. The deoxynucleotides of
DNA are composed of a base constituent (adenine, cytosine, guanine, or thymine), 2ʹdeoxyribose (instead of ribose), and a phosphoryl residue.
Several techniques used in the field of molecular biology are described below.

Polymerase chain reaction (PCR) – This is one of the most important techniques
used in molecular biology and is basically used to copy DNA. PCR allows a single
DNA sequence to be amplified into millions of DNA molecules. PCR can also be
used to introduce mutations within the DNA or introduce special restriction enzyme
sites. In addition, PCR is used to determine whether a certain DNA fragment exists
in a cDNA library. Different types of PCR include reverse transcription PCR (RTPCR) for amplification of RNA and quantitative PCR (QPCR) to measure the
amount of RNA or DNA present.

Expression cloning – This technique helps scientists understand the protein
function. The DNA that codes for a particular protein is cloned or copied using PCR
into an expression vector called a plasmid. The plasmid is introduced to either an
animal cell or a bacterial cell. This plasmid has promoter elements that can stimulate
high expression of the desired protein so that its enzymatic activity can then be
examined.

Gel electrophoresis – This is another important technique used in molecular biology
to separate DNA, RNA, and proteins based on their size by applying an electric field
as the DNA is run through agarose gel.

Macromolecule blotting and probing – Processes such as southern blotting, northern
blotting, western blotting and eastern blotting are used to transfer DNA or RNA
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Applied molecular technique
proteins onto a blotting membrane (often after gel electrophoresis) so they can be
stained or radioactively labelled and then visualized.

Arrays – A DNA microarrays or DNA chip is a collection of DNA spots mounted
on a solid surface such as a microscope slide that can be used to simultaneously
quantify protein expression levels across a large number of genes. The technique
can also be used to genotype various different genomic regions.
Isolation of DNA
Almost every molecular biologist has collected DNA from the organism they are
studying. Initially, isolating DNA was a long and arduous process with large amounts of
DNA collected. Advancing technology has resulted in the amount of DNA needed for
either analysis or cloning of genes to steadily decrease. Nowadays, for example, enough
DNA can be collected for genetic manipulations in laboratory mice or rats from a small
piece of the tail. Human DNA may be analyzed using small blood samples or a few cells
scraped from the inside of the cheek. The decrease in the amount of DNA required for
analysis has allowed scientists to streamline the process so that DNA can be isolated in a
few hours instead of a few days.
Extracting DNA from plants, animals, and bacteria, all require that the cellular
contents be liberated into a solution. Since the bacteria are single cells and contain no
bone, fat, gristle, etc., the DNA is relatively easy to extract. In contrast, samples from
animals and plants must often be ground into tiny fragments before proceeding. Since
plant cells have very rigid cell walls, the scientist must mechanically break the cells open
in a blender, or add special degradative enzymes to digest the cell wall components.
Similarly, to extract DNA from a mouse’s tail, enzymes are added to degrade the
connective tissue and disperse the cells. By far the easiest way to get DNA is to extract it
from bacteria. A few drops of a bacterial culture will give plenty of DNA for most
purposes. First, the bacterial cell wall is easily digested by lysozyme, an enzyme that
degrades the peptidoglycan layer of the cell wall. A successive treatment with detergent
dissolves the lipids of the cell membrane. Chelating agents, such as EDTA (ethylene
diamine tetraacetate), are also used, especially with gram-negative bacteria, to remove
the metal ions that bind components of the outer membrane together. In all these samples,
the cellular contents, including the DNA, are then liberated into solution and are purified
by a further series of steps.
Purification of DNA
Two general types of procedure are used for purification of DNA, centrifugation
and chemical extraction. The principle of centrifugation is as follows. The sample is spun
at high speed and the centrifugal force causes the larger or heavier components to
sediment to the bottom of the tube. For example, destroying the cell wall of bacteria by
lysozyme and detergents leaves a solution containing the fragments of the cell wall, which
are small, and the DNA, which is a gigantic molecule. When the sample is centrifuged,
DNA and some other large components are sedimented to the bottom of the tube. The
fragments of cell wall, together with many other soluble components, remain in solution
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Applied molecular technique
and are discarded. The sedimented DNA is then re-dissolved in an appropriate buffer
solution.
However, it still has a lot of protein and RNA mixed in with it. These are generally
removed by chemical means. One step used in many DNA purifications is phenol
extraction. Phenol, also known as carbolic acid, is very corrosive and extremely
dangerous because it dissolves and denatures the proteins that make up 60 to 70 percent of
all living matter. Consequently, phenol may be used to dissolve and remove all of the
proteins from a sample of DNA.
When phenol is added to water, the two liquids do not mix to form a single solution;
instead, the denser phenol forms a separate layer below the water. When shaken, the two
layers mix temporarily, and the proteins dissolve in the phenol. When the shaking stops,
the DNA solution and phenol containing the proteins separate into two layers. To ensure
that no phenol is trapped with the DNA, the sample is centrifuged briefly. Then the water
containing the DNA and RNA is sucked off and kept. Generally, several successive phenol
extractions are performed to purify away the proteins from DNA.
A variety of newer techniques have been developed that avoid phenol extraction.
Most of these involve purifying DNA by passing it through a column containing a resin
that binds DNA but no other cell components. The two main choices are silica and anion
exchange resins. Silica resins bind nucleic acids rapidly and specifically at low pH and
high salt concentrations. The nucleic acids are released at higher pH and low salt
concentration. Anion exchange resins, such as diethylaminoethyl-cellulose, are positively
charged and bind DNA via its negatively charged phosphate groups. In this case binding
occurs at low salt concentrations and the nucleic acids are eluted by high concentrations of
salt, which disrupt the ionic bonding.
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Applied molecular technique
Removal of Unwanted RNA
Special enzymes remove contaminating RNA from a DNA sample. The enzyme
ribonuclease degrades RNA into short oligonucleotides but leaves the giant DNA
macromolecule unchanged. A mixture of DNA and RNA is first incubated with the
ribonuclease at the optimal temperature for enzyme activity. Next, an equal volume of
alcohol is added. The alcohol precipitates large macromolecules, including long chains of
DNA, out of solution. However, the small RNA fragments remain dissolved. Note that
alcohol treatment is not very specific and will precipitate most large carbohydrates and
many proteins as well as intact macromolecules of both DNA and RNA. Thus, alcohol
precipitation can only be used after these components have been removed from the DNA
by centrifugation and phenol extraction. Next the DNA is sedimented to the bottom of the
tube by centrifugation and the supernatant solution containing the RNA fragments is
discarded. The tiny pellet of DNA left at the bottom of the tube is often scarcely visible.
Nonetheless, it contains billions of DNA molecules, sufficient for most investigations.
This DNA is dissolved into buffered water and is now ready for use in molecular study.
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