Molecular biology

Molecular biology /məˈlɛkjʊlər/ is a branch

of biology that concerns the molecular
basis of biological activity between
biomolecules in the various systems of a
cell, including the interactions between
DNA, RNA, proteins and their biosynthesis,
as well as the regulation of these
interactions.[1]
Writing in Nature in 1961, William Astbury
described molecular biology as:

...not so much a technique as an

approach, an approach from the
viewpoint of the so-called basic
sciences with the leading idea of
searching below the large-scale
manifestations of classical
biology for the corresponding
molecular plan. It is concerned
particularly with the forms of
biological molecules and [...] is
predominantly three-
 dimensional and structural –
which does not mean, however,
that it is merely a refinement of
morphology. It must at the same
time inquire into genesis and
function.[2]

Relationship to other
biological sciences

Schematic relationship between biochemistry genetics

Schematic relationship between biochemistry, genetics
and molecular biology

Researchers in molecular biology use

specific techniques native to molecular
biology but increasingly combine these
with techniques and ideas from genetics
and biochemistry. There is not a defined
line between these disciplines. This is
shown in the following schematic that
depicts one possible view of the
relationships between the fields:[3]

Biochemistry is the study of the

chemical substances and vital
processes occurring in live organisms.
Biochemists focus heavily on the role,
function, and structure of biomolecules.
The study of the chemistry behind
biological processes and the synthesis
of biologically active molecules are
examples of biochemistry.[4]
Genetics is the study of the effect of
genetic differences in organisms. This
can often be inferred by the absence of
a normal component (e.g. one gene).
The study of "mutants" – organisms
which lack one or more functional
components with respect to the so-
called "wild type" or normal phenotype.
Genetic interactions (epistasis) can
often confound simple interpretations of
such "knockout" studies.[5]
 Molecular biology is the study of
molecular underpinnings of the
processes of replication, transcription,
translation, and cell function. The
central dogma of molecular biology
where genetic material is transcribed
into RNA and then translated into
protein, despite being oversimplified,
still provides a good starting point for
understanding the field. The picture has
been revised in light of emerging novel
roles for RNA.[1]

Much of molecular biology is quantitative,

and recently much work has been done at
its interface with computer science in
bioinformatics and computational biology.
In the early 2000s, the study of gene
structure and function, molecular genetics,
has been among the most prominent sub-
fields of molecular biology. Increasingly
many other areas of biology intersecting
with molecular Biology, focus on
molecules, either directly studying
interactions in their own right such as in
cell biology and developmental biology, or
indirectly, where molecular techniques are
used to infer historical attributes of
populations or species, as in fields in
evolutionary biology such as population
genetics and phylogenetics. There is also
a long tradition of studying biomolecules
"from the ground up" in biophysics.[6]

Techniques of molecular
biology

DNA animation

Molecular cloning
Transduction image

One of the most basic techniques of

molecular biology to study protein function
is molecular cloning. In this technique,
DNA coding for a protein of interest is
cloned using polymerase chain reaction
(PCR), and/or restriction enzymes into a
plasmid (expression vector). A vector has
3 distinctive features: an origin of
replication, a multiple cloning site (MCS),
and a selective marker usually antibiotic
resistance. Located upstream of the
multiple cloning site are the promoter
regions and the transcription start site
which regulate the expression of cloned
gene. This plasmid can be inserted into
either bacterial or animal cells. Introducing
DNA into bacterial cells can be done by
transformation via uptake of naked DNA,
conjugation via cell-cell contact or by
transduction via viral vector. Introducing
DNA into eukaryotic cells, such as animal
cells, by physical or chemical means is
called transfection. Several different
transfection techniques are available, such
as calcium phosphate transfection,
electroporation, microinjection and
liposome transfection. The plasmid may
be integrated into the genome, resulting in
a stable transfection, or may remain
independent of the genome, called
transient transfection.[7][8]

DNA coding for a protein of interest is now

inside a cell, and the protein can now be
expressed. A variety of systems, such as
inducible promoters and specific cell-
signaling factors, are available to help
express the protein of interest at high
levels. Large quantities of a protein can
then be extracted from the bacterial or
eukaryotic cell. The protein can be tested
for enzymatic activity under a variety of
situations, the protein may be crystallized
so its tertiary structure can be studied, or,
in the pharmaceutical industry, the activity
of new drugs against the protein can be
studied.[9]

Polymerase chain reaction

Polymerase chain reaction (PCR) is an

extremely versatile technique for copying
DNA. In brief, PCR allows a specific DNA
sequence to be copied or modified in
predetermined ways. The reaction is
extremely powerful and under perfect
conditions could amplify one DNA
molecule to become 1.07 billion molecules
in less than two hours. The PCR technique
can be used to introduce restriction
enzyme sites to ends of DNA molecules,
or to mutate particular bases of DNA, the
latter is a method referred to as site-
directed mutagenesis. PCR can also be
used to determine whether a particular
DNA fragment is found in a cDNA library.
PCR has many variations, like reverse
transcription PCR (RT-PCR) for
amplification of RNA, and, more recently,
quantitative PCR which allow for
quantitative measurement of DNA or RNA
molecules.[10][11]

Gel electrophoresis
Two percent agarose gel in borate buffer cast in a gel
tray.

Gel electrophoresis is one of the principal

tools of molecular biology. The basic
principle is that DNA, RNA, and proteins
can all be separated by means of an
electric field and size. In agarose gel
electrophoresis, DNA and RNA can be
separated on the basis of size by running
the DNA through an electrically charged
agarose gel. Proteins can be separated on
the basis of size by using an SDS-PAGE
gel, or on the basis of size and their
electric charge by using what is known as
a 2D gel electrophoresis.[12]

Macromolecule blotting and

probing

The terms northern, western and eastern

blotting are derived from what initially was
a molecular biology joke that played on the
term Southern blotting, after the technique
described by Edwin Southern for the
hybridisation of blotted DNA. Patricia
Thomas, developer of the RNA blot which
then became known as the northern blot,
actually didn't use the term.[13]

Southern blotting

Named after its inventor, biologist Edwin

Southern, the Southern blot is a method
for probing for the presence of a specific
DNA sequence within a DNA sample. DNA
samples before or after restriction enzyme
(restriction endonuclease) digestion are
separated by gel electrophoresis and then
transferred to a membrane by blotting via
capillary action. The membrane is then
exposed to a labeled DNA probe that has a
complement base sequence to the
sequence on the DNA of interest.[14]
Southern blotting is less commonly used
in laboratory science due to the capacity
of other techniques, such as PCR, to
detect specific DNA sequences from DNA
samples. These blots are still used for
some applications, however, such as
measuring transgene copy number in
transgenic mice or in the engineering of
gene knockout embryonic stem cell
lines.[15]

Northern blotting
Northern blot diagram

The northern blot is used to study the

expression patterns of a specific type of
RNA molecule as relative comparison
among a set of different samples of RNA.
It is essentially a combination of
denaturing RNA gel electrophoresis, and a
blot. In this process RNA is separated
based on size and is then transferred to a
membrane that is then probed with a
labeled complement of a sequence of
interest. The results may be visualized
through a variety of ways depending on
the label used; however, most result in the
revelation of bands representing the sizes
of the RNA detected in sample. The
intensity of these bands is related to the
amount of the target RNA in the samples
analyzed. The procedure is commonly
used to study when and how much gene
expression is occurring by measuring how
much of that RNA is present in different
samples. It is one of the most basic tools
for determining at what time, and under
what conditions, certain genes are
expressed in living tissues.[16][17]
Western blotting

In western blotting, proteins are first

separated by size, in a thin gel sandwiched
between two glass plates in a technique
known as SDS-PAGE. The proteins in the
gel are then transferred to a polyvinylidene
fluoride (PVDF), nitrocellulose, nylon, or
other support membrane. This membrane
can then be probed with solutions of
antibodies. Antibodies that specifically
bind to the protein of interest can then be
visualized by a variety of techniques,
including colored products,
chemiluminescence, or autoradiography.
Often, the antibodies are labeled with
enzymes. When a chemiluminescent
substrate is exposed to the enzyme it
allows detection. Using western blotting
techniques allows not only detection but
also quantitative analysis. Analogous
methods to western blotting can be used
to directly stain specific proteins in live
cells or tissue sections.[18][19]

Eastern blotting

The eastern blotting technique is used to

detect post-translational modification of
proteins. Proteins blotted on to the PVDF
or nitrocellulose membrane are probed for
modifications using specific substrates.[20]
Microarrays

Play media
A DNA microarray being printed

Hybridization of target to probe

A DNA microarray is a collection of spots

attached to a solid support such as a
microscope slide where each spot
contains one or more single-stranded DNA
oligonucleotide fragments. Arrays make it
possible to put down large quantities of
very small (100 micrometre diameter)
spots on a single slide. Each spot has a
DNA fragment molecule that is
complementary to a single DNA sequence.
A variation of this technique allows the
gene expression of an organism at a
particular stage in development to be
qualified (expression profiling). In this
technique the RNA in a tissue is isolated
and converted to labeled complementary
DNA (cDNA). This cDNA is then hybridized
to the fragments on the array and
visualization of the hybridization can be
done. Since multiple arrays can be made
with exactly the same position of
fragments they are particularly useful for
comparing the gene expression of two
different tissues, such as a healthy and
cancerous tissue. Also, one can measure
what genes are expressed and how that
expression changes with time or with
other factors. There are many different
ways to fabricate microarrays; the most
common are silicon chips, microscope
slides with spots of ~100 micrometre
diameter, custom arrays, and arrays with
larger spots on porous membranes
(macroarrays). There can be anywhere
from 100 spots to more than 10,000 on a
given array. Arrays can also be made with
molecules other than DNA.[21][22][23][24]

Allele-specific oligonucleotide

Allele-specific oligonucleotide (ASO) is a

technique that allows detection of single
base mutations without the need for PCR
or gel electrophoresis. Short (20–25
nucleotides in length), labeled probes are
exposed to the non-fragmented target
DNA, hybridization occurs with high
specificity due to the short length of the
probes and even a single base change will
hinder hybridization. The target DNA is
then washed and the labeled probes that
didn't hybridize are removed. The target
DNA is then analyzed for the presence of
the probe via radioactivity or fluorescence.
In this experiment, as in most molecular
biology techniques, a control must be used
to ensure successful
experimentation.[25][26]

SDS-PAGE
In molecular biology, procedures and
technologies are continually being
developed and older technologies
abandoned. For example, before the
advent of DNA gel electrophoresis
(agarose or polyacrylamide), the size of
DNA molecules was typically determined
by rate sedimentation in sucrose
gradients, a slow and labor-intensive
technique requiring expensive
instrumentation; prior to sucrose
gradients, viscometry was used. Aside
from their historical interest, it is often
worth knowing about older technology, as
it is occasionally useful to solve another
new problem for which the newer
technique is inappropriate.[27]

History
While molecular biology was established
in the 1930s, the term was coined by
Warren Weaver in 1938. Weaver was the
director of Natural Sciences for the
Rockefeller Foundation at the time and
believed that biology was about to
undergo a period of significant change
given recent advances in fields such as X-
ray crystallography.[28][29]

Clinical research and medical therapies

arising from molecular biology are partly
covered under gene therapy. The use of
molecular biology or molecular cell biology
approaches in medicine is now called
molecular medicine. Molecular biology
also plays important role in understanding
formations, actions, and regulations of
various parts of cells which can be used to
efficiently target new drugs, diagnose
disease, and understand the physiology of
the cell.[30]

See also
Central dogma of molecular biology
Genetic code
Genome
 Molecular biology institutes
Molecular engineering
Molecular microbiology
Molecular modeling
Protein interaction prediction
Protein structure prediction
Proteome
Bioinformatics
Cell biology

References
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27. Tian, Jie, ed. (2013). Molecular
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Further reading
Cohen, S.N., Chang, A.C.Y., Boyer, H. &
Heling, R.B. Construction of biologically
functional bacterial plasmids in vitro.
Proc. Natl. Acad. Sci. 70, 3240–3244
(1973).
 Rodgers, M. The Pandora's box
congress. Rolling Stone 189, 37–77
(1975).
Keith Roberts, Martin Raff, Bruce
Alberts, Peter Walter, Julian Lewis and
Alexander Johnson, Molecular Biology of
the Cell
4th Edition, Routledge, March, 2002,
hardcover, 1616 pages, 7.6 pounds,
ISBN 0-8153-3218-1
3rd Edition, Garland, 1994, ISBN 0-
8153-1620-8
2nd Edition, Garland, 1989, ISBN 0-
8240-3695-6

External links
 Media related to Molecular biology at
Wikimedia Commons
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