MODULE V

TECHNIQUES IN MOLECULAR BIOLOGY

This module introduces the students to the different techniques or methods used in cell and
molecular biology. Today, classical genetics is often complemented by molecular biology, to
give molecular genetics, which involves the study of DNA and other macromolecules that have been
isolated from an organism. Usually, molecular genetics experiments involve some combination of
techniques to isolate and analyze the DNA or RNA transcribed from a particular gene. In some cases, the
DNA may be subsequently manipulated by mutation or by recombination with other DNA fragments.
Techniques of molecular genetics have wide application in many fields of biology, as well as forensics,
biotechnology, and medicine.

Learning Outcomes:

After examining this module, you are expected to:

Content/Information Sheet:
Molecular Biology methods used to study the molecular basis of biological activity. Most
commonly used methods are protein methods, immunostaining methods, nucleic acid methods. These
methods used to explore cells, their characteristics, parts, and chemical processes, and pays special
attention to how molecules control a cell’s activities and growth.

Molecular Biology Techniques include DNA cloning, cut and paste DNA, bacterial transformation ,
transfection, chromosome integration, cellular screening, cellular culture, extraction of DNA, DNA
polymerase DNA dependent, reading and writing DNA, DNA sequencing, DNA synthesis,
molecular hybridization, rewriting DNA: mutations, random mutagenesis, point mutation, chromosome
mutation. Most important techniques are Polymerase Chain Reaction (PCR), Expression cloning, Gel
electrophoresis, Macromolecule blotting and probing, Arrays (DNA array and protein array).
https://www.omicsonline.org/scholarly/methods-and-techniques-in-molecular-biology-journals-articles-ppts-list.php

Molecular techniques allow the definition of molecular mechanisms and structures that are
responsible for complex processes such as cell growth and division, metabolism, differentiation, and
development. These provide a way to manipulate molecules critical to these processes and observe the
changes in living systems that incorporate the altered molecules. These techniques explain that many
molecular biology laboratory methods take advantage of the simplicity of prokaryotic cell systems such
as bacteria. In prokaryotes, the continuous linear DNA sequence corresponds directly to linear RNA
and protein sequences. However, in eukaryotes, the DNA encoding for protein cannot be read
continuously as it contains interruptions in the translatable sequence. It also describes monoclonal
antibody production techniques, which are used to prepare immunological probes for specific gene
products as well as in situ hybridization, which uses nucleotide probes to localize and study specific
genetic messages in tissue.https://www.sciencedirect.com/science/article/pii/B978044401082750005

I. ISOLATION AND SEPARATION OF NUCLEIC ACIDS

The use of DNA for analysis or manipulation usually requires that it is isolated and purified to
a certain extent. DNA is recovered from cells by the gentlest possible method of cell rupture
to prevent the DNA from fragmenting by mechanical shearing. This is usually in the presence
of EDTA which chelate Mg 2+ ions needed for DNAse (enzymes that degrade DNA).
(https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques)

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 Source: https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques

Figure 1. DNA extraction steps.

ii. Isolation of RNA

The methods used for RNA isolation are very similar to those described above for DNA;
however, RNA molecules are relatively short and therefore less easily damaged by shearing,
so cell disruption can be more vigorous. However, RNA is very vulnerable to digestion by
RNases, which are present endogenously in various concentrations in certain cell types and
exogenously on fingers.

The concentration of the RNA may be estimated by using UV spectrophotometry. At 260 nm,
1 absorbance unit equates to 40 mgml 1of RNA and therefore

40 x A260= concentration of RNA sample (ugml-1)

In RNA a 260 nm:280 nm ratio of approximately 2 would be expected for a sample containing
no contamination.

II. NUCLEIC ACID ELECTROPHORESIS

Electrophoresis in agarose or polyacrylamide gels(PAGE) is the most usual way to separate DNA
molecules according to size (Figure 1.2). The technique can be used analytically or preparative and can be
qualitative or quantitative. Large fragments of DNA such as chromosomes may also be separated by a
modification of electrophoresis termed pulsed field gel electrophoresis (PFGE). The easiest and most
widely applicable method’s electrophoresis in horizontal agarose gels, followed by staining with ethidium
bromide.( https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques)

Electrophoresis is used to check the purity and intactness of a DNA preparation or to assess the
extent of an enzymatic reaction during, for example, the steps involved in the cloning of DNA.

Source: https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques

Figure 2. DNA PAGE set up.

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III. RESTRICTION MAPPING OF DNA FRAGMENTS(RFLP)

Restriction mapping involves the size analysis of restriction fragments produced by several restriction
enzymes individually and in combination.

Source: https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques
Figure 3 . Schematic diagram of restriction fragment length polymorphism(RFLP).

IV. DNA/RNA BLOTTING

The transferring the DNA from the intact gel on to a piece of nitrocellulose or nylon membrane
placed in contact with it-Southern Blotting.
For RNA identification of specific mRNA sequences of a defined length by hybridization to a
labelled gene probe is termed ad Northern Blotting.

Source: https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques
Figure 4 . Blotting step for DNA analysis.

V. POLYMERASE CHAIN REACTION(PCR)

Simple relatively easy of practical manipulation steps using the thermocycler and is the first
techniques used when analyzing DNA and RNA quantitation. It is used to amplify a precise fragment of
DNA from a complex mixture of template DNA and requires little DNA purification. Purified samples will
then be sent to DNA sequencing facilities worldwide to further analyses its phylogenetic relationships.

Supplementary Notes:

Link: https://www.studocu.com/en-gb/document/university-college-london/genetics-development-and-
cancer/lecture-notes-lecture-1-techniques-in-molecular-geneticsdocx/682889

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Summary:

Basic cell and molecular techniques used generally are isolation or extraction of DNA or RNA from tissue
samples. The extracted samples will be amplified via the thermocycler and then confirm for purity by
PAGE or UV spectrophotometry.

Activity:

Create an e-portfolio regarding the different techniques used in cell and molecular biology.

References:

Books:
Karp G. 2005. Cell and Molecular Biology: Concepts and Experiments. 4th ed. John Wiley and Sons, Inc.

Online Resources:

https://scienceprize.scilifelab.se/prize-categories-cell-molecular-biology/

https://www.omicsonline.org/scholarly/methods-and-techniques-in-molecular-biology-journals-
articles-ppts-list.php

https://www.researchgate.net/publication/50364897_Basic_Molecular_Biology_Techniques

https://www.sciencedaily.com/terms/molecular_biology.htm

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