It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase. It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

1. (PCR)
Ajay Subedi
Janamaitri foundation institute of health
sciences,Balaju,Kathmandu
POLYMERASE CHAIN REACTION
1

2. • It was invented in
1983 by Dr. Kary Mullis,
for which he received the
Nobel Prize in
Chemistry in 1993.
• It is called “polymerase” because the only enzyme
used in this reaction is DNA polymerase.
• It is called “chain” because the products of the first
reaction become substrates of the following one, and
so on.
2

3. Why PCR?
1. Simple
2. Powerful
– sensitive
– specific
– reliable
• 3. Fast
3

4. Types of PCR
• 1. Amplified fragment length polymorphism
(AFLP) PCR
• 2. Allele-specific PCR
• 3. Alu PCR
• 4. Assembly PCR
• 5. Asymmetric PCR
• 6. COLD PCR
• 7. Colony PCR
• 8. Conventional PCR
• 9. Digital PCR (dPCR)
• 10. Fast cycling PCR
• 11. High Fidelity PCR
• 12. High-Resolution Melt (HRM) PCR
• 13. Hot start PCR
• 14. In-situ PCR
• 15. Intersequence specific (ISS) PCR
• 16. Inverse PCR
• 17. LATE (Linear-After-The-Exponential)
PCR
• 18. Ligation mediated PCR
• 19. Long-Range PCR
• 20. Methylation-specific PCR (MSP)
• 21. Miniprimer PCR
• 22. Multiplex PCR
• 23. Nanoparticle-Assisted PCR (nanoPCR)
• 24. Nested PCR
• 25. Overlap extension PCR (OE-PCR)
• 26. Real-Time PCR (Quantitative PCR
(qPCR))
• 27. Repetitive sequence-based PCR
• 28. Reverse Transcriptase PCR (RT-PCR)
• 29. Reverse-Transcriptase Real-Time PCR
(RT-qPCR)
• 30. RNase H-dependent PCR
• 31. Single Specific Primer PCR
• 32. Single Specific Primer-PCR (SSP-PCR)
• 33. Solid Phase PCR
• 34. Suicide PCR
• 35. Thermal asymmetric interlaced PCR
(TAIL-PCR)
• 36. Touch down PCR
• 37. Variable Number of Tandem Repeats
(VNTR) PCR
4

5. Principle
• The method relies on thermal cycling, consisting
of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic
replication of the DNA.
• Artificial process which imitates natural DNA
replication
5

6. Materials required for PCR
• PCR tubes
• Tube holder
• Pipettes
• Ice box
• Centrifuge
• PCR machine
• UV transilluminator
6

7. Components of PCR
• DNA template- the sample DNA that contains the target sequence. At the
beginning of the reaction, high temperature is applied to the original
double-stranded DNA molecule to separate the strands from each other.
• Usually target sequence is 100bp-1500bp length
• .DNA polymerase- a type of enzyme that synthesizes new strands of DNA
complementary to the target sequence. The first and most commonly
used of these enzymes is TaqDNA polymerase (fromThermis aquaticus),
whereas PfuDNA polymerase (from Pyrococcus furiosus) is used widely
because of its higher accuracy when copying DNA. Although these
enzymes are subtly different, they both have two capabilities that make
them suitable for PCR:
1) they can generate new strands of DNA using a DNA template and primers,
and
2) they are heat resistant.
7

8. Taq polymerase
• A thermostable DNA polymerase named after
the thermophilic bacterium Thermus aquaticus
• Taq's optimum temperature for activity is 75–
80°C
• Can replicate a 1000 base pair strand of DNA in
less than 10 seconds at 72°C
8

9. • Primers- short pieces of single-stranded DNA that are
complementary to the target sequence. The polymerase
begins synthesizing new DNA from the end of the primer.
-Primers are oligonucleotides with 15-20 bases in length
-Two primers forward and reverse primer .
-Annealing temperature depends upon primer sequence (~
50% GC content)
-Avoid primer complementary (primer- dimer formation)
-The last 3 nucleotides at the 3` end is the substrate for DNA
polymerase - G or C
-The primers must not base pair with each other or with
themselves or form hairpins.
9

10. 10

11. • Nucleotides (dNTPs or deoxynucleotide
triphosphates)- single units of the bases A, T, G, and
C, which are essentially "building blocks" for new
DNA strands.
• Mg or Mn ions- Mg preferred, provides stable
conditions
• Buffer solution-suitable chemical environment for
optimum activity and stability of DNA polymerase
11

12. Very simple Lab procedure
• To perform PCR, the extracted sample (which
contains target DNA template) is added to a
tube containing primers, free nucleotides
(dNTPs), and Taq polymerase. The PCR
mixture is placed in a PCR machine. PCR
machine increases and decreases the
temperature of the PCR mixture in automatic,
programmed steps which generates copies of
the target sequence exponentially.
12

13. Thermocycling protocol
annealing
95ºC 95ºC
55-60ºC
72ºC
4ºC
5 min 1 min
30 sec
1 min
∞ Hold
Initial denaturation
of DNA
1X 35-40X 1X
extension
denaturation
13

14. 14

15. 15

16. 16

17. Initial Denaturation
• Heating separates the double
stranded DNA
– Denaturation
– heating the reaction to a
temperature of 94–96 °C for 5
mins depending on the GC
content of the template
• performed only once at the
beginning of the reaction
Heat Cool
17

18. Denaturation
• First regular cycling event and consists of
heating the reaction to 94–98 °C for 30–60
seconds.
• Causes melting of the DNA template by
disrupting the hydrogen bonds between
complementary bases, yielding single-
stranded DNA molecules.
18

19. Annealing (primer binding)
• Annealing of the primers to the
single-stranded DNA template.
• Typical annealing temperature is
between 50 and 55°C, for 30-60
seconds
• optimal temperature depends on
the primer sequence and length
• Typically the annealing temperature
is about 3-5 degrees Celsius below
the Tm of the primers used.
• The polymerase binds to the
primer-template hybrid and begins
DNA formation.
19

20. Extension(synthesis of new DNA)
o DNA polymerase duplicates DNA by synthesizing a new DNA
strand complementary to the DNA template strand (72C)-optimal
temperature for Taq DNA polymerase
o During this step dNTPs that are complementary to the template in
5' to 3' direction are added
20

21. Extension Temperature
Target sequence Length Time
≤ 500bp 30 seconds
500 - 1500bp 60 seconds
>1500bp 90 seconds
21

22. Final extension
• This single step is occasionally performed at a
temperature of 70–74 °C for 5–15 minutes
after the last PCR cycle
• Its objective is to ensure that any remaining
single-stranded DNA is fully extended.
22

23. Final Hold
• This step at 4°C for an indefinite time may be
employed for short-term storage of the
reaction.
23

24. PCR Amplification
Exponential Amplification of template DNA
24

25. Detection of PCR products
Agarose Gel electrophoresis
 easiest and commonest way to separate and analyze DNA on the
basis of molecular weight
 have lower resolving power for DNA than acrylamide gels, but they
have greater range of separation, and are therefore usually used for
DNA fragments of 50-20,000 bp in size
 the lower the concentration of the gel, the larger the pore size, and
the larger the DNA that can be sieved
-technique consist of 3 basic steps
• preparation of agarose
• Electrophoresis of DNA fragments
• Visualization of DNA fragments
25

26. Preparation of Agarose Gel
• Insoluble in water and buffer at RT but
dissolves on boiling
• On cooling undergoes polymerization (sugar
polymers crosslink with each other causing
solution to gel)
• Density or pore size determined by the
concentration of agarose
• 0.5 -2% agarose use, usually 1% preferred
• Buffer- 1x TBE(tris-borate-EDTA) or TAE
26

27. Electrophoresis of DNA
• Technique used to separate charged molecules
• DNA is negatively charged at neutral PH
• DNA migrates to anode when electric field is
applied across the gel
• Migration of DNA depends upon
-molecular size of DNA
-agarose concentration
-applied current
-conformation of DNA
27

28. • Progress of gel is monitored by observing the
migration of visible dye-tracking dye
• Commonly used are Xylene cynol and
Bromophenol blue
• 50 volts for 1-2 hrs
28

29. Visualization of DNA
• DNA is not visible in gel
• Stained with specific dye such as ethidium
bromide
-1 µl of 10 mg/ml
• Visualize under UV light-DNA floresces
*ethidium bromide must be handled carefully as it
is mutagen and carcinogen
29

30. 30

31. PCR phases
 Exponential
◦ If 100% efficiency – exact doubling of products. Specific and
precise
 Linear
◦ High variability. Reaction components are being consumed
and PCR products are starting to degrade.
 Plateau
◦ End-point analysis. The reaction has stopped and if left for
long – degradation of PCR products.
31

32. 32

33. 33
REAL TIME PCR
(RT-PCR)

34. Lets see a short animation
video on Lab procedure of RT-
PCR for diagnosis of COVID-19.
34

36. Real time PCR
• In order to amplify small amounts of DNA, the
same methodology is used as in conventional
PCR using a DNA template, at least one pair of
specific primers, deoxyribonucleotides, a
suitable buffer solution and a thermo-stable
DNA polymerase.

37. Principal:
• Quantitative PCR is carried out in a thermal
cycler with the capacity to illuminate each
sample with a beam of light of a specified
wavelength and detect the fluorescence
emitted by the excited fluorophore.
• The thermal cycler is also able to rapidly heat
and chill samples, thereby taking advantage of
the physicochemical properties of the nucleic
acids and DNA polymerase.

38. • A substance marked with a fluorophore is
added to this mixture in a thermal cycler that
contains sensors for measuring the
fluorescence of the flurophore after it has been
excited at the required wavelength allowing
the generation rate to be measured for one or
more specific products.
38

39. • This allows the rate of generation of the amplified product
to be measured at each PCR cycle. The data thus generated
can be analysed by computer software to calculate relative
gene expression (or mRNA copy number) in several
samples.
• This measurement is made after each amplification cycle,
and this is the reason why this method is called real time
PCR (that is, immediate or simultaneous PCR) not at its
end, as in conventional PCR. In the case of RNA
quantitation, the template is complementary DNA (cDNA),
which is obtained by reverse transcription of ribonucleic
acid(RNA). In this instance the technique used is
quantitative RT-PCR or Q-RT-PCR.

40. • Two common methods for the detection of
products in quantitative PCR are:
- Non-specific fluorecent dyes that intercalate with
any double-stranded DNA
- Sequence-specific DNA probes consisting of
oligonucleotides that are labelled with a fluorescent
reporter which permits detection only after
hybridization of the probe with its complementary
sequence to quantify messenger RNA (mRNA) and
non-coding RNA in cells or tissues.

41. 41

42. Advantages
• Real-Time chemistries allow for the detection of PCR
amplification during the early phases of the reaction.
• Measuring the kinetics of the reaction in the early phases of
PCR provides a distinct advantage over traditional PCR
detection. Traditional methods use Agarose gels for
detection of PCR amplification at the final phase or end-
point of the PCR reaction
• Sensitive assay, highly quantitative, and highly reproducible
• Can detect as few as 5 molecules
• Good Excellent dynamic range, linear over several orders of
magnitude
• Useful for diagnostic purposes

43. Disadvantages of Real time PCR:
• Expensive
• Can pick up RNA carryover or contaminating RNA leading to
false positives
• Need a skillful person

45. Medical applications OF PCR
• genetic testing, where a sample of DNA is
analyzed for the presence of genetic
disease mutations. Prospective parents can be
tested for being genetic carriers, or their
children might be tested for actually being
affected by a disease.
• Prenatal testing can be obtained
by amniocentesis,chorionic villus sampling, or even
by the analysis of rare fetal cells circulating in
the mother's bloodstream. PCR analysis is also
essential to preimplantation genetic diagnosis,
where individual cells of a developing embryo are
tested for mutations.
45

46. Medical applications:
• PCR can also be used as part of a sensitive test
for tissue typing, vital to organ transplantation.
• blood type
• Oncogenes.
46

47. • Characterization and detection of infectious disease
organisms have been revolutionized by PCR:
• The human immunodeficiency virus
• tuberculosis,
• The spread of a disease organism through populations
• The sub-types of an organism that were responsible
for earlier epidemics can also be determined by PCR
analysis.
47

48. Forensic applications
• In its most discriminating form, genetic
fingerprinting can uniquely discriminate any one
person from the entire population of the world.
• DNA database of earlier evidence or convicts.
Simpler versions of these tests are often used
to rapidly rule out suspects during a criminal
investigation. Evidence from decades-old
crimes can be tested, confirming the people
originally convicted.
• parental testing, where an individual is
matched with their close relatives.
48

49. In Molecular diagnosis
• Viral load and identification of virus
• Bacterial infection: early detection specific targeting and better
treatment
detect Mycobacterium tuberculosis
Legionella pneumophila,
Listeria monocytogenes,
Neisseria gonorrhoea.
• Antibiotic resistance :
Staphylococcus aureus,
Staphylococcus epidermidis,
Helicobacter pylori,
Enterococcus faecalis
Enterococcus faecium