PCR PRINCIPLES

1. POLYMERASE CHAIN
REACTION
DR. S. SELVA MANI
SECOND YEAR POST GRADUATE
DEPT OF PUBLIC HEALTH DENTISTRY

2. CONTENTS
• Introduction
• What is PCR?
• History of PCR
• Basic Requirements
• Essential Components of PCR
• Principles of PCR
• Instrumentation
• RT-PCR
• Dental Applications of PCR
• Advantages of PCR
• Limitations of PCR
• Conclusion
• References

3. GENOME
•A genome is an organism’s complete set of DNA, including all of its
genes.
•Each genome contains all of the information needed to build and
maintain that organism.
•In humans, a copy of the entire genome—more than 3 billion DNA
base pairs—is contained in all cells that have a nucleus
The first whole genome to be
sequenced was of the
bacterium Haemophilus influenzae
The worm Caenorhabditis elegans was the
first animal to have its whole genome
sequenced.

4. •Genome sequencing is figuring out the order of DNA nucleotides,
or bases, in a genome—the order of As, Cs, Gs, and Ts that make up
an organism's DNA.
•The human genome is made up of over 3 billion of these genetic
letters. Machines "read" a sequence of DNA bases.
•A DNA sequence that has been translated from life's chemical
alphabet into our alphabet of written letters might look like this.
GENOME SEQUENCING

5. • Genome sequence will represent a valuable shortcut, helping
scientists find genes much more easily and quickly
GENOME SEQUENCING
•The whole genome can't be sequenced all at once because available
methods of DNA sequencing can only handle short stretches of DNA
at a time.
•Break the genome into small pieces, sequence the pieces, and then
reassemble them in the proper order to arrive at the sequence of the
whole genome -TYPES 1."clone-by-clone" approach
• 2. "whole-genome shotgun" method
HOW TO DO GENOME SEQUENCING?

6. HOW DOES THE SEQUENCING MACHINE KNOW
WHETHER A BASE IS AN A, C, G, OR T?
• DNA been chopped up, copied, chemically modified, and tagged with
fluorescent dyes corresponding to the four different DNA bases, or
genetic letters.
•A piece of DNA is copied many times,- then divided into four batches
-another round of copying.
•In this second round, a small amount of chemically modified base is
added to each batch—that is, modified T to one batch, A to another,
and so on.. The result of all this is that one batch of DNA will contain
only pieces that end in T, another only pieces that end in A, a third only
pieces that end in G, and the fourth batch only pieces that end in C

7. The Human Genome Project was an international research effort to determine the
sequence of the human genome and identify the genes that it contains. The Project was
coordinated by the National Institutes of Health and the U.S. Department of Energy
The main goals of the Human Genome Project were to provide a complete and accurate
sequence of the 3 billion DNA base pairs that make up the human genome and to find all of
the estimated 20,000 to 25,000 human genes.

8. WHAT IS PCR?
•Polymerase chain reaction (PCR) is technique for
generating large quantities of a specified DNA.
•PCR is a cell free amplification technique for synthesizing
multiple identical copies of any DNA of interest.
•Purpose of the PCR is to amplify the lot of double stranded
DNA molecules(fragments) with same size and sequence by
enzymatic method and cycling condition.

9. POLYMERASE CHAIN REACTION
POLYMERASE CHAIN REACTION

10. SHORT HISTORY OF PCR
• 1983: Dr. Kary Mullis developed PCR
• 1985: First publication of PCR by
Cetus Corporation appears in Science.
• 1986: Purified Taq polymerase is first used in PCR
• 1988: PerkinElmer introduces the automated
thermal cycler.
• 1989: Science declares Taq polymerase "molecule of the
year.

11. SHORT HISTORY OF PCR
• 1990: amplification and detection of specific DNA
sequences using a fluorescent DNA-binding dye, laying
the foundation for future "real-time" or "kinetic" PCR.
• 1991: RT-PCR is developed using a single
thermostable polymerase, rTth, facilitating
diagnostic tests for RNA viruses.
• 1993:Dr. Kary Mullis shares Nobel Prize in
Chemistry for conceiving PCR technology.

12. SHORT HISTORY OF PCR
• 1999: Dynal launches DRB-36 HLA-typing kit for
tissue typing.
• 2003: HIV-1 MONITOR Test, version 1.5 Product
Family
• AMPLICOR® CT/NG Test for Chlamydia
trachomatis,
• AMPLICOR® CT/NG Test for Neisseria gonorrhoeae

13. BASIC REQUIREMENTS FOR
PCR REACTION
1) DNA sequence of target region must be
known.
2) Primers - typically 20-30 bases in size. These
can be readily produced by commercial
companies. Can also be prepared using a DNA
synthesizer

14. BASIC REQUIREMENTS FOR
PCR REACTION
• 3) Thermo-stable DNA polymerase - eg Taq
polymerase which is not inactivated by heating
to 95C
4) DNA thermal cycler - machine which can be
programmed to carry out heating and cooling of
samples over a number of cycles.

15. ESSENTIAL
COMPONENTS
• DNA template
• DNA polymerase
• Primer dNTPs
• Buffer to maintain PH
• Divalent cations (Mg+2)
• Monovalent cation Potassium ions

17. CONDITION
1. DENATURATION OF DS DNATEMPLATE
2. ANNEALING OF PRIMERS
3. EXTENSION OF DS DNA MOLECULES

18. REQUIREMENTS FOR PCR
•Template extracted DNA is the identified target sequence that
requires to be amplified. DNA polymerase is a key enzyme to
replicate target sequences of DNA that attaches individual nucleotides
together to generate the PCR product.
• Primer molecules are appropriate short and single-stranded
sequences of DNA or RNA designed to particularly anneal to a
desired nucleic acid target.
•Forward and reverse primer pairs have a length of 18–22 base pairs.

19. REQUIREMENTS FOR PCR
•For PCR amplification, DNA is extracted from a desired target and
added to the reaction mix tube including primers, PCR buffer,
deoxynucleotides (dNTP), MgCl2 and DNA polymerase enzyme in an
examination tube.
•The reaction tube then located in a thermocycler that runs repeated
cycles of DNA replication to take place in the following steps.

20. DENATURATION
Denaturation of DNA– Heating reaction tube to 94°C for
separation of the double strands DNA and yielding two
single strands of DNA molecule

21. ANNEALING
•At 50–65°C, forward and reverse primers, anneal to a
particular site at each of the single-stranded DNA
templates. Tm of the primer pairs determines the
annealing temperature

22. EXTENSION/ELONGATION
Extension -at 72°C, new complementary DNA strands
synthesizes by the elongation of primers using DNA
polymerase enzyme

23. STAGES OF
PCR

24. CYCLING

25. PCR STEP
AND
CYCLE

26. CYCLE -I
The new DNA strand joined to each primer is beyond
the sequence that is complementary to second primer.
New strands are referred as long template.
They will be used in second cycle.

27. CYCLE -II
• The DNA strands (original & newly synthesized long template) are
denatured, annealed with primers & subjected to DNA synthesis.
•At the end of second round, long templates & short templates (DNA
strands with primer sequence at one end & sequence complementary
to other end primer) are formed.

28. CYCLE -III
•The original DNA strands along with long & short
templates are starting materials.
•Denaturation, renaturation & synthesis are repeated.
• This process is repeated again & again for each cycle.
•At the end of 32nd cycle of PCR, about million fold target
DNA is synthesized.

30. TYPES OF PCR
•Real-time PCR
•Quantitative real time PCR
(Q-RT PCR)
•Reverse Transcriptase PCR
(RT-PCR)
•Multiplex PCR
•Nested PCR
•Long-range PCR
•Single-cell PCR
•Fast-cycling PCR
•Methylation-specific PCR (MSP)
•Hot start PCR
•High-fidelity PCR
•In situ PCR
•Variable Number of Tandem
Repeats (VNTR) PCR
•Asymmetric PCR
•Repetitive sequence-based PCR
•Overlap extension PCR
•Assemble PCR

31. INSTRUMENTATION

36. REVERSE TRANSCRIPTASE -PCR
•In RT-PCR, complementary DNA (cDNA) is made by reverse
transcribing of the RNA templates with the enzyme reverse
transciptase.
•This technique is used to qualitatively study gene expression, and
can be combined with real time PCR (qPCR) to quantify RNA
levels.
•RT-PCR requires an RNA template, enzyme, nucleotides, buffers
and thermocyclers to produce RT-PCR products. Kits are available to
simplify and streamline the process.

50. PERIODONTOLOGY
•Parra and Slots studied the association of human viruses in the GCF
of persons with different types of periodontal disease and reported
the prevalence of DNA viruses such as (HSV), 2 (EBV IRUS).
•Ssygunet al. showed CMV in crevicular specimens of chronic
periodontal wounds frequently exists and indicated a strong
correction between HCMV and EBV-1 in sub gingival regions with
deep furrows along with a loss of insertion.
•Q-PCR offers a sensitive, efficient, and accurate method for
quantification of genes expression. Lyons and coworkers used
TaqMan® systems for determining the quantity of P. gingivalis and
the entire numbers of bacteria exist in bacterial plaque.

51. PERIODONTOLOGY
•Commercial diagnostic tests (e.g., MyPerioPath® , MicroDent®
Test, ParoCheck® kits, and oral DNA® ) using multiplex PCR are
obtainable to assess the microbiota in subgingival plaque samples
and they give important information for a prevention approach for
healthy people and therapy strategies for “at risk” patients

52. DENTAL CARIES
•Rupfet and coworkers used from a competitive PCR technique for
the particular quantitative evaluation of S. mutans. PCR allowed a
rapid and precise evaluation of unknown amounts of S. mutans and
provided an efficient tool for observing the performance of
therapeutic and preventative procedures as well as estimating the risk
of dental caries in patients .
•Glucosyl transferase-directed PCR is available for private practices
of routine screening for S. mutans by plaque and saliva (LCL
Biokey)

53. DENTAL CARIES
•Kukletová isolated and taxonomically characterized lactobacilli
inhabiting rampant caries in children and use rep-PCR fingerprinting
method for characterization of Lactobacillus spp. related with dental
caries.
•Saarela and coworkers studied power of PCR method in the
differentiation of S. sobrinus and S. mutans serotypes and lineages,
they proved that PCR is suitable method for differentiating S. mutans
lineages and epidemiological research .

54. ENDODONTIC INFECTIONS
•Saito and coworkers employed a q-PCR procedure in primary
endodontic infections based on single copy genes. Their result
displayed the highly prevalence and abundant of T. forsythia but
moderate frequency and less abundant of P. gingivalis in the study
groups.
•Baumgartner and coworkers used PCR method to evaluate cellulite
aspirations and abscesses of infected root canals for the presence of
Candida albicans. Their results show that PCR is a very sensitive
molecular technique and can be utilized to directly detect C. albicans
in endodontic infection

55. ENDODONTIC INFECTIONS
•For example, Jafari and coworkers assessed the antibacterial
efficacy of photodynamic and 2.5% NaOCl treatment against E.
faecalis-infected root canals by means of Q-PCR method .
IMPLANT RELATED INFECTIONS
•Trampuz et al. using Multiplex PCR diagnosed the periprosthetic
joint infection (PJI) in sonication fluid from removed dental
implants &assessment for who formerly took antibiotics.
•Multiplex PCR has the potential for more improvement of the PJI
diagnosis with modified primer sets

56. PERI-IMPLANTITIS
•r avoid the risk of peri-implantitis, this method plays a role in
identifying bacteria causing peri-implantitis before implant
placement. Real time PCR has identified opportunistic
microorganisms such as E. faecalis in peri-implant location of
diseased implants suggesting elimination of prosthesis and routine
decontamination of implant surface and implant abutment connection
GENETIC POLYMORPHISM
•Modified gene on chromosome 11 which result in a loss in cathepsin C
function and development of Papillon–Lefevre syndrome.
•Genetic polymorphisms in MPO-463G/A gene and Fc gamma receptor
gene were evaluated by means of RT-PCR and allele-specific PCR,

57. ORAL CANCER PATHOGENESIS
•The one recent study revealed human papilloma virus genomic DNA
in approximately 26% of all Head and neck squamous cell carcinoma
by sensitive PCR-based.
•Sand et al. investigated clinically healthy oral mucosa, oral
leukoplakia, and oral squamous cell carcinoma (OSCC) for presence
of HSV-1 via nested PCR method.
•Chiao-Wen et al. used QPCR to analyze 6 SNPs of CD44 in oral
cancer patients and healthy controls. The CD44 rs187115
polymorphism has prognostic importance in oral cancer and also may
be used as factors to predict the clinical stage in oral cancer patients

58. INFLAMMATORY MARKERS
•By means of real time PCR expression level of receptor activator of
NF-KB ligand (RANKL) and matrix metallo proteinases was found
to be associated with expression of interleukin-1β, TNF-α, IF-γ.
•Using semiquantitative PCR gene expression of RANKL to
osteoprotegerin (OPG) ratio was found to be increased in
periodontitis.
•mRNA expression level of several growth factors such as toll-like
receptors (TLRs), NALP3 and NOD2 as well as signaling mediators
CD14, MYD88, and TIR-domain-containing adapter-inducing
interferon-beta (TRIF) were evaluated by RT-PCR technique

59. RECENT ADVANCES
•Gene microarray technology or “global expression profiling” is
based on the ability to deposit numerous different DNA strands on a
small surface, typically a chip.
•The different DNA sequences are organized in columns and rows
such that the identity of each sequence is recognized through its
position on the array.
•Methods like RT-PCR allow analyzing for only a few genes per
sample. But microarray technology not only analyses more genes
than was feasible formerly, but also can observed genes are not
influenced by preselection of genes.

60. ADVANTAGES OF PCR
• Simplicity of quantification, high sensitivity, accuracy, rapid
analysis, reproducibility, quality control, and minimum
contamination
• Accurate detection of strains of different microorganisms with
different phenotype.
• Enormous samples can be evaluated at one time
• RT-PCR is a sensitive technique for finding of viruses and mRNA
expression levels.
• Real time-PCR has the capability to evaluate the actual number of
targets existing in the clinical samples

61. ADVANTAGES OF PCR
• Nested PCR simplifies the identification of bacterial DNA present
at very low levels.
• Multiplex PCR has the capability to examination for variety of
organisms or genes in one reaction tube
• Study of strictly anaerobic microorganisms, in which cell death
could happen during sampling and transportation, where cell
viability is not important factor in PCR method.

62. LIMITATIONS
•Required to high level of expertise.
•Annealing of primers to the similar sequences of the template DNA
could be changed specificity of nonspecific amplified PCR product.
The DNA polymerase enzyme utilized in the PCR technique is prone
to errors which can cause mutations product DNA molecules.
•Mixing different primers in multiplex PCR can cause some
interfering in amplification.
•In the Q-PCR, the fluorescent signal cannot distinguish nonspecific
versus specific PCR products.
•When nested PCR -detecting DNA of periodontal microorganisms,
numerous false negative results -contaminate other reaction tubes

63. CONCLUSION
• PCR is not only vital in the clinical laboratory by amplifying
small amounts of DNA for detection, but it is also important for
genetic predisposing for defects such as Factor V Leiden.
• The PCR technology can also be employed in law enforcement,
genetic testing of animal stocks and vegetable hybrids, and drug
screening along with many more areas.

64. REFERENCES
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