4. MOLECULAR BIOLOGY TECHNIQUE
1.1. Molecular biology technique
Introduction
Molecular biology is the study of biology at a molecular
level (eg. Replication, transcription & translation of the
genetic material)
Molecular biology chiefly concerns itself with understanding
the interactions between the various systems of a cell,
including the interactions between DNA, RNA & protein
biosynthesis & learning how these interactions are
regulated
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5. MOLECULAR BIOLOGY TECHNIQUE
1.2. Technique purpose & basic procedure
Technique used in Molecular Biology
Microscopic observation
Centrifugation
Extraction
Electrophoresis
Chromatography
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6. MOLECULAR BIOLOGY TECHNIQUE
1.2.1 Microscopic observation
1. Microscopic Observation
Microscope: instrument designed to produce magnified
visual or photographic images of objects too small to
be seen with the naked eye
The microscope must accomplish three tasks:
(a) produce a magnified image of the specimen
(b) separate the details in the image
(c) render the details visible to the human eye or camera
Multiple-lens (compound microscopes) designs with
objectives & condensers
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7. MOLECULAR BIOLOGY TECHNIQUE
1.2.1 Microscopic observation
Some common types of microscopes which can be
used in the study of cells are
(a) Light (optical) microscopes
(b) Phase contrast microscopes
(c) Transmission electron microscope
(d) Scanning electron microscope
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8. MOLECULAR BIOLOGY TECHNIQUE
1.2.1 Microscopic observation
Phase contrast microscope
Many cell details cannot be seen using an ordinary
optical microscope. This is because there is very little
contrast between structures. They have similar
transparency & are not coloured
Special phase contrast condensers & objective lenses
are added to the light microscope.
Light rays travelling through material of different
densities are bent & altered giving a better contrast.
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9. MOLECULAR BIOLOGY TECHNIQUE
1.2.1 Microscopic observation
Phase contrast microscopes enable living, non-
pigmented specimen to be studied without fixing &
staining
This type of microscope give better contrast but do not
improve resolution
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10. MOLECULAR BIOLOGY TECHNIQUE
1.2.1 Microscopic observation
Electron microscope (EM)
Uses an electron beam instead of light rays
Electrons have short wavelengths ( ~ 0.0005 nm).
This give a high resolving power to the EM which can
resolve two objects that are only ~ 1 nm apart
Electrons are negatively charged & can be focussed
by the use of electromagnets in the EM.
Magnification range from 15x to 200,000x
There are two main types of EM: Transmission EM &
Scanning EM
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11. MOLECULAR BIOLOGY TECHNIQUE
1.2.1 Microscopic observation
Transmission electron microscope (TEM)
- study the ultra-structure of a cell
Scanning electron microscope (SEM)
- produce 3-dimentional view of objects
- eg. cells, tissue & small organism
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12. MOLECULAR BIOLOGY TECHNIQUE
1.2.2 Centrifugation
2. Centrifugation
A piece of equipment, generally driven by a motor, that
puts an object in rotation around a fixed axis, applying a
force perpendicular to the axis.
The centrifuge works using the sedimentation principle
(separate substances or greater & lesser density)
There are many different kinds of centrifuges, including
those for very specialised purposes.
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14. MOLECULAR BIOLOGY TECHNIQUE
1.2.3 Extraction
3. Extraction
Molecules that can be extracted are:
(a) DNA
(b) RNA
(c) protein
DNA extraction is a routine procedure to collect DNA for
subsequent molecular or forensic analysis
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16. MOLECULAR BIOLOGY TECHNIQUE
1.2.4 Electrophoresis
4. Electrophoresis
Is a technique used to separate substances with
different charges
Eg. Proteins in an electric field
Other mixture include amino acids & nucleic acid
fragments especially DNA fragments for fingerprinting
The medium used can be paper, gel layer or in a
column
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17. MOLECULAR BIOLOGY TECHNIQUE
1.2.4 Electrophoresis
Electrodes are placed on both end of wet paper or gel
on a piece of glass
In agarose gel electrophoresis, DNA and RNA can be
separated on the basis of size by running the DNA
through an agarose gel
Proteins can be separated on the basis of size by
running the DNA through an 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 change by using what is known as a 2D gel
electrophoresis
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1.2.4 Electrophoresis
Functions
Very useful to separate proteins, as they are
delicate. Enzymes separated by this technique are still
active.
To diagnose diseases as when blood plasma
proteins are separated, extra proteins found could be
antibodies (Ab) formed to combat certain pathogens.
The Ab are compared with standard ones & extracted
to determine the actual type
For DNA-fingertyping, which is used to identify
individual in forensic science
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19. MOLECULAR BIOLOGY TECHNIQUE
1.2.4 Electrophoresis
Gel electrophoresis
DML 202 General Biology
& Human Genetics
11/16/2011 19
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(Chapter 17: Molecular
20. MOLECULAR BIOLOGY TECHNIQUE
1.2.4 Electrophoresis
Principle of gel electrophoresis. Influence of charge and particle size on the
electrophoretic mobility of proteins or other macromolecules like nucleic
acids. A. Separation by charge, B. Separation by particle size, C: Addition
of A and B, D: Compensation of A and B.
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21. MOLECULAR BIOLOGY TECHNIQUE
1.2.4 Electrophoresis
Limitations
Only small amounts of substance can be separated
Substances which are of no charge or too similar in
charges cannot be separated
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22. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
5. Chromatography
Is a technique used to separate mixtures of chemicals of
similar nature (eg. photosynthesis pigments) by allowing
their common solvent flowing over them in a solid medium
such as paper
Can separate other mixtures, which include proteins, amino
acids, nucleic acids, nucleotides, fatty acid,
monosaccharides & disaccharides
The solid media are paper, gel layer or column of
cellulose & achrimide polymer
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23. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Types of Chromatography
Paper chromatography
Two dimensional paper chromatography
Thin-layered chromatography
Column chromatography
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24. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
A. Paper chromatography
Initially, a small but concentrated amount of mixture e.g leaf extract
is applied on one end of the paper. The paper is hung on a common
solvent such as petroleum ether.
When the solvent goes up, the solute will separate as indicated by
the different
coloured spots
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26. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
B. Two dimensional paper chromatography
Can be done in 2 dimensions with a square piece of
paper when there are too many solutes in the mixture.
Is done1st with one solvent then the paper is turned
90° to be separated with another solvent giving a
better separation such as with a mixture of amino
acids
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28. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Principle
It involves passing a mixture dissolved in a “mobile
phase” through a stationary phase, which separates
the analyte to be measured from other molecules in the
mixture and allows it to be isolated.
Methods used to separate and/or to analyze complex
mixtures based on differences in their structure and/or
composition
The components to be separated are distributed
between two phases: a stationary phase bed and a
mobile phase which percolates through the stationary
bed
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29. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Test molecules which display tighter interactions with
the support will tend to move more slowly through the
support than those molecules with weaker interactions
Even very similar components, such as proteins that
may only vary by a single amino acid, can be separated
with chromatography
Repeated sorption/desorption acts that take place
during the movement of the sample over the stationary
bed determine the rates. The smaller the affinity a
molecule has for the stationary phase, the shorter the
time spent in a column
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30. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Rf
Important character of the solute in a certain solvent
The ratio of the distance moved by the solute to that
moved by the solvent
Distance moved by the solute
Rf =
Distance moved by the solvent
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31. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Rf is a constant used to determine the position of an
unknown solute if the Rf under the same condition is
known
Used to identify an unknown spot in the chromatogram
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32. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Functions
The technique is simple & can be easily carried out to
separate chemicals of similar nature.
It takes a short time to carry out. A simple separation of
leaf pigments only takes less than 30 minutes
It requires only simple apparatus such as paper and
dropper to apply the mixture on the paper
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33. MOLECULAR BIOLOGY TECHNIQUE
1.2.5. Chromatography
Limitations
Only small amounts of substances can be separated at
one time
When the solutes are too similar like certain amino
acids, they are not separable by this technique
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