Brief overview of PCR-SSCP, rationale, principle, protocol and applications

1. PCR-SSCP(Single Strand Conformation
Polymorphism)
Presented to:
•Dr. Atif Kamran
Presented by:
• Fatima Iftikhar MS-19
Session:
• 2020-2022
• University of the Punjab

2. Rationale
PCR-SSCP (single-strand conformation
polymorphism) analysis is the easiest and one of the
most sensitive mutation detection technique.
This technique has been widely used for the detection
of mutations.
This technique has also been useful for detecting
polymorphisms that are linked to various genes.
1

3. While the
sensitivity of
PCR-SSCP
tends to decrease
with increasing
fragment length.
PCR-SSCP
analysis is much
more sensitive to
the replication
errors that occur
during the PCR.
In PCR-SSCP
analysis,
changes in
several hundred
bases are
detected.
DETECTION
2

4. More
laboratory
skills are
required to
optimize it.
When the main
purpose of a
particular
genotyping
experiment is
the detection of
an unknown
SNP, the best
technique is
PCR SSCP.
The concept of
PCR-SSCP is
based on the
presence or
absence of a
particular
mutation
between
normal and
mutant
amplicons.
3

5. How PCR-SSCP works?
The main concept of PCR-SSCP is based on
the initial separation (melting) of the double-
stranded forms by heat into single stranded
forms.
In the separated state, the molecules tend to
fold into a three-dimensional conformation
according to their nucleic acid sequences.
In the polyacrylamide gel electrophoresis, the
separated single strands of DNA molecules
accommodate different three-dimensional
structures with different mobilities. 4

6. Structural
conformations are
affected by a
mutation at a
particular
nucleotide
position in the
primary sequence,
which can alter
the physical
conformation of
the denatured
single strand
bands.
This alteration
often leads to the
slightly tilted
positioning of the
mutant single-
strand bands with
respect to their
normal
counterparts in
the neutral
polyacrylamide
gels.
5

7. For better results
amplified products
should be less than
300 bp.
PCR-SSCP can be
made high-
throughput and
automated by using
fluorescent labelled
primers .
6

8. 7

9. Applications
It is very easy, safe, fast and
for this reason widely applied
in clinical diagnostic, forensic
medicine, biochemical,
veterinary, microbiological,
food and environmental
laboratories.
One of the possible
applications of the
method is the diagnosis
and identification of
mutations in new
strains of
coronaviruses.
It is used for the
detection of somatic
mutations in cancer.
8

10. Animals
( e.g:
Escherichia
coli in
the black swan,
egg production
traits in hens )
Birds
Genes
identification
in ostrich popu
lation
Fishes
Identification
of shrimp speci
es.
Plants
Genetic diversity
of citrus trees,
identification of
genetic diversity
of lemon varieties
Food and
Forensic
Microbial
communities
identification
in cheese
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11. References
Al-Shuhaib, M. B. S., Al-Lamy, S. M., Al-Tayy, H. M., Al-Thuwaini, T. M., & Radhi, A. H. (2018). Single
nucleotide polymorphism (SNP) of leptin gene in Holstein cattle. Thai Journal of Veterinary Medicine, 48(2),
187-201.
Battilani, M., Coradin, T., Scagliarini, A., Ciulli, S., Ostanello, F., Prosperi, S., & Morganti, L. (2003).
Quasispecies composition and phylogenetic analysis of feline coronaviruses (FCoVs) in naturally infected cats.
FEMS Immunol Med Microbiol, 39, 141-147.
Csikos, A., Hodzic, A., Pasic-Juhas, E., Javor, A., Hrković-Porobija, A., Goletic, T., & Czegledi, L. (2016).
Applicability and sensitivity of PCR-SSCP method for milk species identification in cheese. Acta
Alimentaria, 45(1), 69-76.
Gasser, R. B., Hu, M., Chilton, N. B., Campbell, B. E., Jex, A. J., Otranto, D., & Zhu, X. (2006). Single-strand
conformation polymorphism (SSCP) for the analysis of genetic variation. Nature protocols, 1(6), 3121-3128.
Hayashi, K. (1992). PCR-SSCP: a method for detection of mutations. Genetic Analysis: Biomolecular
Engineering, 9(3), 73-79.
Kakavas, K.V. (2021). Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism
method. Mol Biol Rep, 48, 3629-3635. 10