This document discusses several molecular techniques used in microbiology, including restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), and cleaved amplified polymorphic sequences (CAPS). RFLP detects genetic variations by cutting DNA with restriction enzymes and comparing fragment lengths. PFGE improves on standard gel electrophoresis by applying an alternating electric field to better separate very large DNA molecules. CAPS combines PCR and RFLP to detect single base changes without radioactive probes. These techniques are useful for genome mapping, disease analysis, and forensic or epidemiological investigations.

1. Molecular Techniques in Microbiology
Keezia Khurshid
Ph.D. Scholar (AAHM)
Unit V

2. Restriction Fragment Length Polymorphism
RFLP is a variation in the length
of a DNA fragment produced by
a specific restriction enzyme
acting on a DNA from different
individuals that usually results
from a genetic mutation.
The principle of RFLP markers is
that any genomic DNA can be
differentiated according to the
presence or absence of
restriction enzyme sites.
A restriction enzyme cuts the
DNA molecule at every
occurrence of a particular
sequence, called restriction
sites.

4. 1. Isolation of DNA:
Isolation of DNA is the first step for many DNA based
technologies. DNA is found either in nuclear chromosomes or
in organelles (mitochondria and chloroplast).
To extract the DNA from its location, several laboratory
procedures are needed to break the cell wall and nuclear
membrane, and so approximately separate the DNA from other
cell components.

5. 2. Restriction Digestion & Gel Electrophoresis
Extracted DNA is
digested with specific
restriction enzymes.
Each restriction enzyme
will recognize and cut
DNA in a predictable
way under appropriate
conditions resulting in a
reproducible set of
DNA fragments
(restriction fragments)
of different lengths.
The millions of restriction
fragments are then
separated by gel
electrophoresis, because
the fragments would be
seen as a continuous
smear if stained with
ethidium bromide,
staining alone cannot
detect the polymorphism.
Hybridization, therefore
must be used to detect
specific polymorphism.

6. 3. DNA transfer by Southern Blotting
This technique is named
after E.M. Southern.
In this method the gel is
first denatured in a basic
solution and placed in a
tray. A porous nylon or
nitrocellulose membrane
is laid over the gel.
All the DNA restriction
fragments in the gel
transfer as single strand
will retain the same
pattern on the
membrane as on the gel
due to capillary action.

7. 4. DNA hybridization & visualization
The labelled RFLP probe is hybridized with DNA
on the nitrocellulose paper.
The RFLP probes are complimentary as well as
labelled with radioactive isotopes so they form
color band under visualization by autoradiography.

9. Advantages of RFLP:
Produces semi-dominant markers, allowing determination of homozygosity or
heterozygosity.
Stable and reproducible, gives constant results over time and location
No prior information on DNA sequence is required.
Relatively simple technique.

10. Limitations of RFLP:
Very long
methodology, before
results are gained.
High labor
requirements.
High quality and
large quantities of
DNA must be used.
Must frequently
work with
radioisotopes.
Many probes are not
available depending
on species.
To many
polymorphism may
be present for a
short probe.
Cost of
development is very
high due to time,
and labor
requirements.

11. Application of RFLP:
1. Genome mapping: 2. Genetic disease analysis:

12. 3. Paternity test
4. Criminal investigation

13. Cleaved Amplified Polymorphic Sequence
CAPS is also
known as PCR-
RFLP.
It was
developed after
the emerge of
PCR.
It was originally
conceived as a
technique to
detect base
changes in DNA
sequences and
therefore acts as
a tool in the
diagnosis of
genetic diseases.
It was soon
recognized that
the technology
would have
much wider
applications in
genetic diversity
studies.
It is an
alternative to
direct
sequencing for
detecting
sequence
variation.

15. Advantages:
Most CAPS markers are co-
dominant and locus-specific.
Most CAPS genotypes are
easily scored and interpreted.
CAPS markers are easily
shared between laboratories.
CAPS assay does not require
the use of radioactive
isotopes, and it is more
amenable, therefore, to
analyses in clinical settings.

16. Disadvantages:
Sequence the RFLP probe.
Design primers to amplify 800–2,000-bp DNA fragments. Targeting introns or 3' untranslated
regions should increase the chance of finding polymorphisms
The PCR product is cloned and sequenced.
PCR amplify DNA fragments from target genotypes, separately digest the amplicons with one
or more restriction enzymes.
Screen the digested amplicons for polymorphism on gels stained with ethidium bromide.

17. Restriction Enzymes:

18. Mechanism of Action
• Restriction Enzymes recognize a specific sequence of
nucleotides, and produce a double stranded cut in the DNA.
• These cuts are of two types:
• Blunt Ends
• Sticky Ends

19. Type
I
Type
II
Type
III
Types of Restriction Enzymes

20. Electrophoresis
It is a technique used for
the separation of DNA,
RNA or protein molecules
using an electric field
applied to gel matrix.
The most common
technique for this
purpose is that of
standard agarose gel
electrophoresis.

21. SAGE technique was
unable to separate very
large molecules of DNA
effectively.
David C. Schwartz and
Charles Cantor, 1984
developed a variation on
the standard protocol by
introducing an alternating
voltage gradient to
improve the resolution of
larger molecules.
This technique was later
on known as Pulse Field
Gel Electrophoresis
(PFGE).

22. Pulse Field Gel Electrophoresis
Pulsed Field Gel Electrophoresis (PFGE) is a
technique used for the separation of large
DNA molecules by applying to a gel matrix
an electric field that periodically changes
direction.
Principle: with periodic changing of field
direction, the various lengths of DNA react to
the change at differing rates. That is, larger
pieces of DNA will be slower to realign their
charge when field direction is changed, while
smaller pieces will be quicker.

23. Major steps involved:

24. Procedure

25. Applications
Since, field gel electrophoresis allows the separation of DNA
fragments containing up to 100,000 bp (100 kilobase pairs,
or kbp), characterization of such large fragments has allowed
construction of a physical map for the chromosomes from
several bacterial species.
PFGE may be used for genotyping or genetic
fingerprinting.
It is commonly considered a gold standard in epidemiological
studies of pathogenic organisms.
Subtyping has made it easier to discriminate among strains
of Listeria monocytogenes and thus to link environmental or
food isolates with clinical infections.

26. Advantages

27. Limitations