Polymerase Chain Reaction (PCR) is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample in the presence of DNA polymerase, primers, and nucleotides. Each cycle doubles the amount of target DNA. After 20-30 cycles, there can be over a billion copies of the original DNA sequence. PCR is used for a variety of applications including disease diagnosis, cloning genes, forensic analysis, and more. It is a powerful technique that has revolutionized molecular biology.

1. Polymerase Chain Reaction
Manjunath S Hurakadli
Department of Plant Pathology
UAS GKVK Bengaluru - 65

2. Diagnosis of viral diseases
• Biological indexing- Live test plants, controlled green
house, labour intensive and time consuming
• Serological methods- ELISA, DIBA, Dip strips
• Molecular methods- Dot blot, PCR/ RT PCR

3. P
urine
P y r im id in e
Guanine
Adenine
Cytosine Thymine Uracil
Molecular techniques are based on the structure of DNA and RNA

4. Thymine Adenine
Cytosine
Guanine
Adenine
Guanine
Thymine
Cytosine

5. What is PCR?
PCR is an exponentially progressing synthesis of the defined target
DNA sequences in vitro.
 PCR, “discovered” in 1983 by Kary Mullis, enables the
amplification (or duplication) of millions of copies of any
DNA sequence with known flanking sequences.
Requires only simple, inexpensive ingredients and a couple hours.
Can be performed by hand or in a machine called a thermal cycler.
1993: Nobel Prize for Chemistry

6. What is PCR? :
Why “Polymerase”?
It is called “polymerase” because the
only enzyme used in this reaction is
DNA polymerase.

7. What is PCR? :
Why “Chain”?
It is called “chain” because the
products of the first reaction become
substrates of the following one, and
so on.

8. What is PCR? :
The “Reaction” Components
1) Target DNA - contains the sequence to be amplified.
2) Pair of Primers - oligonucleotides that define the sequence
to be amplified.
3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.
4) Thermostable DNA Polymerase - enzyme that
catalyzes the reaction
5) Mg++ ions - cofactor of the enzyme
6) Buffer solution – maintains pH and ionic strength
of the reaction solution suitable for the activity of
the enzyme

9. • Typical PCR Reaction
• 12.5 μl dH2O
• 2 μl 10 X PCR buffer + mg
• 1 μl 200 μM dNTP
• 1 μl 50 μM Left Primer
• 1 μl 50 μM Right Primer
• 2 μl Template DNA
• 0.5 μl Taq Pol (5 Units/ μl)
20 μl Total Vol

10. Master Mix
• 1 volume master mix
– 32.5 μl dH2O
– 5 μl 10 X PCR buffer
– 1 μl 200 μM dNTP
– 0.5 μl Taq Pol (5 Units/ μl)
39 μl Total Volume

11. PCR Buffer
• Basic Components
– 20mM Tris-HCL pH 8.4
– 50mM KCl
– 1.5 mM MgCl2
• Magnesium – Since Mg ions form complexes with dNTPs, primers and
DNA templates, the optimal concentration of MgCl2 has to be selected
for each experiment. Too few Mg2+ ions result in a low yield of PCR
product, and too many increase the yield of non-specific products and
promote misincorporation.
• Potential Additives
– Helix Destabilisers - useful when target DNA is high G/CWith NAs of
high (G+C) content.
• dimethyl sulphoxide (DMSO),
• dimethyl formamide (DMF),
• urea
• formamide
– Long Targets >1kb. Formamide and glycerol
– Low concentration of template: Polyethylene glycol (PEG)

12. Primers
• Paired flanking primers
• Length (17-28bp)
• GC content 50-60%
• GC Clamp
• Tm’s between 55-80
• Avoid simple sequences – e.g. strings of G’s
• Avoid primer self complementary
– e.g. hairpins, homodimers, heterodimers

13. Hot water bacteria:
Thermus aquaticus
Taq DNA polymerase
Life at High Temperatures
by Thomas D. Brock
Biotechnology in Yellowstone
© 1994 Yellowstone Association for Natural Science
http://www.bact.wisc.edu/Bact303/b27

14. Heat-stable DNA Polymerase
• Given that PCR involves very high temperatures, it is
imperative that a heat-stable DNA polymerase be used in the
reaction.
• Most DNA polymerases would denature (and thus not
function properly) at the high temperatures of PCR.
• Taq DNA polymerase was purified from the hot springs
bacterium Thermus aquaticus in 1976
• Taq has maximal enzymatic activity at 75 C to 80 C, and
substantially reduced activities at lower temperatures.

15. PCR Polymerases
• Taq, Tht, Thf. Pfu, others
• Native or Cloned
• Fidelity (Error Rate)
– Taq 1/10,000nt, Pfu 1/1,000,000

16. The three main steps of PCR
• The basis of PCR is temperature changes and the effect that these
temperature changes have on the DNA.
• In a PCR reaction, the following series of steps is repeated 20-40 times
(note: 25 cycles usually takes about 2 hours and amplifies the DNA
fragment of interest 100,000 fold)
Step 1: Denature DNA
At 95C, the DNA is denatured (i.e. the two strands are separated)
Step 2: Primers Anneal
At 40C- 65C, the primers anneal (or bind to) their complementary
sequences on the single strands of DNA
Step 3: DNA polymerase Extends the DNA chain
At 72C, DNA Polymerase extends the DNA chain by adding nucleotides to
the 3’ ends of the primers.

17. Denaturation of DNA
This occurs at 95 ºC mimicking the function of
helicase in the cell.

18. Step 2 Annealing or Primers Binding
Primers bind to the complimentary sequence on the
target DNA. Primers are chosen such that one is
complimentary to the one strand at one end of the
target sequence and that the other is complimentary
to the other strand at the other end of the target
sequence.
Forward Primer
Reverse Primer

19. Step 3 Extension or Primer Extension
DNA polymerase catalyzes the extension of the
strand in the 5-3 direction, starting at the
primers, attaching the appropriate nucleotide
(A-T, C-G)
extension
extension

20. • The next cycle will begin by denaturing the
new DNA strands formed in the previous
cycle

21. Overview of PCR
1. Temperature Cycling
Denaturation 94°
Annealing 55°
Extension 72°
2. Every cycle DNA between
primers is duplicated

22. Fig. 9.3
Denature
Anneal PCR Primers
Extend PCR Primers
w/Taq
Repeat…

23. How PCR works:
1. Begins with DNA containing a sequence to be amplified and a pair of synthetic
oligonucleotide primers that flank the sequence.
2. Next, denature the DNA to single strands at 94˚C.
3. Rapidly cool the DNA (37-65˚C) and anneal primers to complementary single-
straned sequences flanking the target DNA.
4. Extend primers at 70-75˚C using a heat-resistant DNA polymerase such as Taq
polymerase derived from Thermus aquaticus.
5. Repeat the cycle of denaturing, annealing, and extension 20-45 times to produce 1
million (220) to 35 trillion copies (245) of the target DNA.
6. Extend the primers at 70-75˚C once more to allow incomplete extension products
in the reaction mixture to extend completely.
7. Cool to 4˚C and store or use amplified PCR product for analysis.

24. Exponential Amplification
30 cycles --- 1 billion copies in theory

26. Primer annealing
5'
5'
3'
3'
target DNA
repeat PCR cycles
5'
5'
3'
3' Double-stranded DNA
Denaturation
5'
5'
3'
3'
Extension
3'
5'
5'
5'
5'
3'
3'
3'
3'
5'
5'
5'
5'
3'
3'
3'
Extension
PCR Round 1
DNA polymerase always adds nucleotides to
the 3’ end of the primer

27. denaturation
primer annealing
extension
PCR Round 2
Chromosomal strand
Long strand
After the second round of
PCR, the number of long
strands increases
arithmetically and the
number of short strands
increases exponentially (the
number of chromosomal
strands is always the same).
5'
5'
3'
3'
5'
5'
3'
3'
3'
5'
5'
5'
5'
3'
3'
3'
3'
5'
5'
5'
5'
3'
3'
3'
3' 5'
3'
5'
5'
3'
5'
5'
3'
3'
3'
5'
5'
3'
Short strand

28. 72 0C - primer extension
94 0C - denaturation
Temperature 0C
Temperature control in a PCR thermocycler
94 0C - denaturation
50 – 70 0C - primer annealing

29. # PCR cycles
After 25 cycles have 3.4 x 107 times more DNA
plateau is reached after
25-30 cycles

30. A PCR product should be confirmed in at least two ways initially.
These can include:
1. Correct product size.
2. Sequence the product.

31. Amplified DNA on Agarose gel electrophoresis
Intensity of PCR product depends on concentration of targeted DNA

32. How pure is your sample?
The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for
ssRNA. Ratios lower than 1.7 usually indicate significant
protein contamination.
The A260/A230 ratio of DNA and RNA should be roughly
equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower
ratios may indicate contamination by organic compounds
(e.g. phenol, alcohol, or carbohydrates).

33. Running your sample through an agarose
gel is a common method for examining the
extent of DNA degradation.
Good quality DNA should migrate as a high
molecular weight band, with little or no
evidence of smearing.
genomic
DNA
RNA
(degraded)
Checking for Degradation: DNA/RNA

34. RT-PCR amplification of OYDV in garlic
PRSV
Chi8
Jap10
Chi11
Chi4
Chi10
Jap2
Chi5
Chi1
Jap1
Isrl
Jap8
Chi12
Chi3
Chi2
Chi7
Jap9
Jap7
Jap4
Netd
Jap11
Chi9
Chi6
Brazil
Gdel
Jap6
Jap5
Jap12
Jap3
100
100
67
84
56
100
100
69
83
65
90
62
100
55
90
100
53

35. Primer Design Considerations
• Primers must be specific for desired sequence to be amplified
– primers should be long enough to ensure specificity
(usually 18-30 bases)
– primers normally screened against databases
• Primers must form stable duplex at annealing temperature
• No complementarity between forward and reverse primers or
primers and product

36. Initial primer selection criteria
• Length (18-25 bases)
• Base composition (45-55% GC)
• Melting temperature (55-80C)
• 3’ terminal sequence
– strong bonding base (G or C) at end

37. Primer complementarity criteria
• Primer vs. self & forward vs. reverse
– maximum number of consecutive bonds
– maximum number of consecutive G-C bonds
• Forward primer vs. Reverse primer
– maximum number of consecutive bonds between the 3’
ends
• Primer vs. product
– maximum number of consecutive bonds between the 3’
ends

38. Optimization criteria
• Melting temperatures should be similar for
both primers
• Product should be as short as allowable

39.  Amplify DNA for Cloning (PCR)
 Amplify DNA for sequencing without cloning (PCR)
 DNA sequencing reaction (PCR)
 Mapping genes and regulatory sequences
 Linkage analysis (identify genes for traits/diseases)
 Diagnose disease
 Pathogen screening
 Sex determination
 Forensic analysis
 Paternity/maternity (relatedness)
 Behavioral ecology studies (relatedness)
 Molecular systematics and evolution (comparing homologous sequences in
different organisms)
 Population genetics (theoretical and applied)
 Physiological genetics (studying basis of adaptation)
 Livestock pedigrees (optimize breeding)
 Wildlife management (stock identification/assessment)
 Detection of Genetically Modified Food (GMOs)
Applications of PCR

40. Conclusion
The speed and ease of use, sensitivity, specificity and
robustness of PCR has revolutionised molecular biology and
made PCR the most widely used and powerful technique with
great spectrum of research and diagnostic applications.

41. Thank you

42. Higher GC content has higher thermal stability while
lower GC content has low thermostability. Meaning a DNA
with more GC content is highly stable due to the presence of
more hydrogen bonds, though research shows that the
hydrogen bonds do not have a direct impact on the stability of
the DNA.

43. CP
TCAGGAGGAAACCCAACACCGCCGCCAGTTGTGGATGCTGGCGCGGACACTAGCAAGGA
TAAGAAAGAGAAAAGCAACAGGGGAAAAGGCCCTGAAAGTTCTGAAGGGTCAGACAACAA
CAACCGTGGAACAGAGAATCAGTCAATGAGAGATAAGGATGTGAATGCTGGTTCCAAAGG
AAAAGTGGTTCCTCGGCTACAAAGGATCACAAAAAGGATGAATTTGCCAATGGTGAGAGG
AAATGTGATCTTGAACTTAGATCATCTGTTAGATTACAAGCCAGAACAAACTGATCTTTTCA
ACACAAGAGCAACAAAGATGCAGTTTGAAATGTGGTACAATGCTGTGAAGAGCGAGTATG
AAATAGATGATGAACAGATGTCAATTGTAATGAATGGCTTTATGGTGTGGTGTATTGATAAT
GGCACTTCACCAGATGTGAATGGTACTTGGGTAATGATGGATGGAGAAGAGCAAGTGGAA
TATCCACTCAAACCAATGGTTGAAAATGCAAAGCCAACACTCCGCCAGATCATGCACCATT
TCTCAGATGCAGCTGAAGCATACATTGAGATGAGAAATTCTGAGAAGCCGTACATGCCTAG
GTACGGATTACTTCGGAATTTGAGGGACAAAAATCTAGCTCGCTACGCTTTTGATTTCTAT
GAAGTGACATCCAAAACGTCGGATCGAGCAAGAGAAGCAGTAGCACAGATGAAGGCAGC
AGCCCTCAGCAACGTTAACAGCAAGTTGTTTGGACTTGATGGTAATGTGGCAACAACCAG
CGAGAATACTGAAAGGCACACTGCAAGGGATGTTAATCAAAACATGCACACACTTCTTGGC
ATGGGCTCCCCGCAG