PCR

1. Vivekanandha Arts and Science College for
Women, sankari Salem
Submitted by
Sabitha Govindan
III-BSC Microbiology
Department of Microbiology
Vivekananda arts and science
college for women
TOPIC: PCR TECHNOLOGY

4. CONTENT
● History
● Definition
● Principle of PCR
● Steps involved in PCR
● Components of PCR
● Basic requirements of PCR
● Types of PCR
● Applications of PCR
● Advantages of PCR
● Disadvantages of PCR
● Conclusion

5. HISTORY
● 1983 Dr.Kary mullis developed PCR.
● Developed in 1983 by Kary Mullis, PCR is now a common technique
used in clinical and research laboratories for a broad variety of
applications.
● In 1993, Mullis was awarded the Nobel Prize in chemistry for his
work on PCR.
DEFINITION
● PCR is a technique that takes specific sequence of DNA of small
amount and amplifies it to be used for further testing.
● Polymerase chain reaction (PCR) is a technique used in molecular
biology to amplify a single copy or a few copies of a segment of DNA
across several orders of magnitude, generating thousands to millions
os copies of a particular DNA sequence.

7. PRINCIPLE OF PCR
● The PCR technique is based on the enzymatic replication of
DNA. In PCR, a short segment of DNA is amplified using primer
mediated enzymes.
● DNA synthesis new strands of DNA complementary to the
template DNA. The DNA polymerase can add a nucleotide to the
pre-existing 3’-OH group only.
STEPS INVOLVED IN PCR
Denaturation
● Temperature: 92 - 94°c
● Double stranded DNA melts into single stranded DNA
Annealing
● Temperature ~50 - 70°c( dependent on the melting temperature
of the expected duplex)

8. ● Primer bind to their complementary sequences.
EXTENSION
● Temperature: ~72°c
● Time : 0.5 - 3 minutes
● DNA polymerase binds to their annealed primers and extends DNA
at the 3’ end of the chain
COMPONENTS OF PCR
● DNA Template
● DNA polymerase
● Oligonucleotide primers
● De oxyribonucleotide triphosphate
● Buffer system

9. BASIC REQUIREMENTS FOR PCR REACTION
● DNA sequence of target region must be known.
● Primers - 20-30 bases in sizes.
● These can be readily produced by commercial companies. Can be
prepared using a DNA synthesizer.
● Thermostable DNA polymerase.
● DNA thermal cycler - machine which can be programmed to carry
out heating and cooling of samples over a number of cycles.

10. TYPES OF PCR
Real time PCR
● In this type, the DNA amplification is detected in real-time
with the help of a fluorescent reporter. The signal strength of
the fluorescent reporter is directly proportional to the number
of amplified DNA molecules.
Nested PCR
● This was designed to improve sensitivity and specificity. They
reduce the non-specific binding of products due to the amplification
of unexpected primer binding sites.

11. Multiplex PCR
● This is used for the amplification of multiple targets in a single
PCR experiment. It amplifies many different DNA sequences
simultaneously.
Quantitative PCR
● It uses the DNA amplification linearity to detect, characterize
and quantify a known sequence in a sample.
Arbitrary primed PCR
● It is a DNA fingerprinting technique based on PCR. It uses
primers the DNA sequence of which is chosen arbitrarily.

12. ▣ DNA Template
▣ DNA polymerase
▣ Oligonucleotide primers
▣ De oxyribonucleotide triphosphate
▣ Buffer system
● The following are the applications of PCR :
● Testing of genetic disease mutations.
● Monitoring the gene in gene therapy.
● Detecting disease-causing genes in the parents.
● Used as a tool in genetic fingerprinting.
● Identifying the criminal from millions of people.
● Paternity tests
MEDICINE
APPLICATION OF PCR
FORENSIC SCIENCE

13. RESEARCH AND GENETICS
● Compare the genome of two organisms in genomic
studies.
● In the phylogenetic analysis of DNA from any source
such as fossils.
● Analysis of gene expression.
● Gene Mapping

14. 1. Highly specific: PCR can distinguish DNA sequences by just
one nucleotide, making it a very accurate technique.
2. Sensitive: PCR is a very useful technique when the amount of
DNA sample is limited because it allows the detection of even a
single copy of a specific DNA template.
3. Versatile: The PCR technique can be used for various
applications like genetic testing, criminal investigations, and
paternity tests.
4. Rapid and efficient: PCR can efficiently and rapidly amplify a
small amount of DNA sample to million copies in just a few hours.
ADVANTAGES OF PCR

15. ● 1. Contamination: The PCR technique is very susceptible to
contamination from other sources of DNA or RNA or the
environment. This can mislead data interpretation.
● 2. Cost and complexity: PCR can be expensive and requires
expert knowledge for high-throughput projects.
● 3. Lack of novel information: Since PCR can only amplify and
target specific DNA sequences targeted by the primers, PCR
provides limited information and cannot detect novel DNA
sequences.
DISADVANTAGE OF PCR

16. ● Errors in amplification: Base substitutions, indels, and
other alterations in DNA sequences can lead to
inaccurate amplification and hence, false results.
● Inhibition from sample content: the whole PCR cycle can
be disrupted by inhibitors that co- purify with DNA, such
as heme from blood samples, reducing the sensitivity of
the process.

19. CONCLUSION
● PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA for STD detection,
but it also important for genetic predisposing for
defects such as factor leiden
● PCR technology can also be employed in law
enforcement, genetic testing of animals stocks and
vegetable hybrids and drug screening along with many
more areas.