1. Polymerase Chain Reaction & its applications
Dr.Shubha Gopal
Associate Professor & Chairperson
Department of Studies in Microbiology
University of Mysore
Manasagangotri
Mysore –
UGC Academic Staff College

3. The Polymerase Chain Reaction (PCR) was not a discovery, but rather an invention
A special DNA polymerase (Taq) is used to make many copies of a short length of DNA (100-10,000 bp) defined by primers
Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry

5. Development….
PCR work was first published (1985)using Klenow polymerase – unstable with heat
New enzyme had to be added manually at each step
Maximum length 400bp
Great idea – not very practical
First reports using DNA polymerase
from Thermus aquaticus (1988)
Taq-polymerase (Saiki et al, 1988) from
Yellow stone National Park hot springs
Developed automatic “thermocycler” programmable heat block

7. Polymerase Chain Reaction (PCR)
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

8. PCR Thermocycler

9. What all PCR Can Do ?
Starting with one original copy an almost infinite number of copies can be made using PCR
“Amplified” fragments of DNA can be sequenced, cloned, probed or sized using electrophoresis
Defective genes can be amplified to diagnose any number of illnesses
Genes from pathogens can be amplified to identify them (i.e., HIV, Vibrio sp., Salmonella sp. etc.)
Amplified fragments can act as genetic fingerprints

10. PROCEDURE …..

11. PCR Reagents 1X Buffer MgCl2 dNTPs Primers DNA polymerase DNA
10mM Tris-HCl, 50mM KCl
MgCl2
1mM - 4mM (1.5mM)
dNTPs
200μM
Primers
100nM-1μM, 200nm (or less) for real time analysis
DNA polymerase
Taq DNA polymerase is thermostable
1-4 Units (1 unit)
DNA
10pg-1μg (20ng)

12. Different types of buffers

14. Polymerase Chain Reaction

16. Initiation - Forming the Replication Eye
Origin of Replication 5’ 3’ 5’ 3’ 3’ 5’ 5’ 3’

17. Extension - The Replication Fork5’ 3’
Primase
Single strand binding proteins
Laging Strand 3’ 5’ 5’ 3’
Okazaki fragment
RNA Primers
DNA Polymerase
Helicase
Leading Strand 5’

18. How are the functions of replication achieved during PCR ???
ENZYMES
Helicase
SSB proteins
Topoisomerase
DNA pol
Primase
Ligase
. Heat
Melting DNA
Polymerizing DNA
. Taq Polymerase
Providing primer
. Primers added to the reaction mix
. N/A as fragments are short
Joining nicks

19. PCR
Temperature
100 50
T i m e
Melting
94 oC 5’ 3’

20. PCR
Temperature
100 50
T i m e
Melting
94 oC 5’ 3’
Heat 5’ 3’

21. PCR T i m e Temperature Melting Melting 94 oC 94 oC Extension
100 50
T i m e
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC 5’ 3’ 5’ 3’ 5’

22. PCR T i m e Heat Heat 30x Temperature Melting 94 oC Extension
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100 50
T i m e
30x 5’ 3’
Heat 5’ 5’
Heat 5’ 3’ 5’

23. PCR T i m e 30x Temperature Melting 94 oC Extension Annealing Primers
100 50
T i m e
30x 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 5’

24. PCR T i m e Heat Heat 30x Temperature Melting 94 oC Extension
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100 50
T i m e
30x 3’ 5’ 5’
Heat 5’ 5’
Heat 5’

25. PCR T i m e 30x Temperature Melting 94 oC Extension Annealing Primers
100 50
T i m e
30x 3’ 5’ 5’ 5’

26. PCR T i m e 30x Temperature Fragments of defined length Melting 94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100 50
T i m e
30x 3’ 5’
Fragments of defined length 5’ 5’

27. More Cycles = More DNA
Number of cycles
Size
Marker

28. PCR Optimisation 1: Buffers
Most buffers have only KCl (50mM) and Tris (10mM)
Concentrations of these can be altered
KCl facilitates primer binding but concentrations higher than 50mM inhibit Taq
DMSO, BSA, gelatin, glycerol, Tween-20, Nonidet P-40, Triton X-100 can be added to aid in the PCR reaction
Enhance specificity, but also can be inhibitory
Pre-mixed buffers are available

29. PCR Optimisation 2: MgCl2
MgCl2: required for primer binding
MgCl2 affects primer binding, Tm of template DNA, product- and primer-template associations, product specificity, enzyme activity and fidelity
dNTPs, primers and template chelate and sequester the Mg ion, therefore concentration should be higher than dNTPs (as these are the most concentrated)
Excess magnesium gives non-specific binding
Too little magnesium gives reduced yield

30. PCR Optimisation 3: Primer Design
Specific to sequence of interest
Length nucleotides
Annealing temperature 50oC-70oC
Ideally 58oC-63oC
GC content 40-60%
3’ end critical (new strand extends from here)
GC clamp (G or C at 3’ terminus)
Inner self complementarity:
Hairpins <5, dimers <9
3’ complementarity:
<3-4 bases similar to other primer regions

31. PCR Optimisation 4: Cycling Conditions
Denaturation:
Some Taq polymerases require initial denaturation (hot start)
Annealing temperature:
~ 5oC less than Tm of primers
Tm = 4(G + C) + 2(A + T)oC (or use of primer software)
Decrease in annealing temperature result in non-specific binding
Increase in annealing temperature result in reduced yield

32. PCR Optimisation 5: Cycle Number
25-40 cycles
Half-life of Taq is 30 minutes at 95oC
Therefore if you use more than 30 cycles at denaturation times of 1 minute, the Taq will not be very efficient at this point
Theoretical yield = 2n
ie. cycle 1 = 2, cycle 2 = 4, cycle 3 = 8, etc
eg. if you start with 100 copies after 30 cycles you will have 107, 374, 182, 400 copies

33. In summary
Primer length should not exceed 30 mer.
Tm, not more than 60 degree .
GC Content should be in the range of % for optimum PCR efficiency.
Primers should end (3′) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming.

34. GCG PRIME
The GCG program PRIME is a good tool for the design of primers for PCR and sequencing
For PCR primer pair selection, you can choose a target range of the template sequence to be amplified
In selecting appropriate primers, PRIME allows you to specify a variety of constraints on the primer and amplified product sequences.
upper and lower limits for primer and product melting temperatures
primer and product GC contents.
a range of acceptable primer sizes
a range of acceptable product sizes.
required bases at the 3' end of the primer (3' clamp)
maximum difference in melting temperatures between a pair of PCR primers

35. PC Software
There are a number of (expensive) dedicated PCR primers design programs for personal computers that have “special features” such as nested and multiplex PCR :
Oligo (Molecular Biology Insights, Inc.)
Primer Premier (Premier Biosoft)
Many of the comprehensive MolBio. programs also have PCR features
Mac Vector
OMIGA
Vector NTI
Gene Tool

36. Primer Problems primers should flank the sequence of interest
primer sequences should be unique
primers that match multiple sequences will give multiple products
repeated sequences can be amplified - but only if unique flanking regions can be found where primers can bind

37. PCR Based Methods Sequence Specific Oligonucleotide (SSO) probe
Amplified fragment-length polymorphism to generate finger prints
Large VNTR regions (10-30 b.p. repeat)
Short Tandem Repeats (STR) (2-7 b.p. repeat)
RAPD using universal primers
Rep- PCR (ERIC primers)
PCR- Ribotyping (16S rDNA regions)

38. Variations of the PCR Colony PCR Nested PCR Multiplex PCR AFLP PCR
Hot Start PCR
In Situ PCR
Inverse PCR
Asymmetric PCR
Long PCR
Long Accurate PCR
Reverse Transcriptase PCR
Allele specific PCR
Real time PCR

39. Types of PCR
Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA segments cloned. Nested PCR: Involves two consecutive PCR reactions of 25 cycles. The first PCR uses primers external to the sequence of interest. The second PCR uses the product of the first PCR in conjunction with one or more nested primers to amplify the sequence within the region flanked by the initial set of primers. Inverse PCR: Used to amplify DNA of unknown sequence that is adjacent to known DNA sequence. Quantitative PCR: Product amplification w r t time, which is compared with a standard DNA. Hot start PCR: Used to optimize the yield of the desired amplified product in PCR and simultaneously to suppress nonspecific amplification.

40. Colony PCR
Colony PCR- the screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products.
Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube.
Heat the mix in a boiling water bath (90-100C) for 2 minutes
Spin sample for 2 minutes high speed in centrifuge.
Transfer 20 μl of the supernatant into a new microfuge tube
Take 1-2 μl of the supernatant as template in a 25 μl PCR standard PCR reaction.

41. Hot Start PCR
This is a technique that reduces non-specific amplification during the initial set up stages of the PCR
The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase
DNA Polymerase- Eubacterial type I DNA polymerase, Pfu
These thermophilic DNA polymerases show a very small polymerase activity at room temperature.

42. Nested PCR
Two pairs (instead of one pair) of PCR primers are used to amplify a fragment.
First pair -amplify a fragment similar to a standard PCR. Second pair of primers-nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.
Advantage- Very low probability of nonspecific amplification

44. Multiplex PCR
 Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers.

45. Inverse PCR
Inverse PCR (Ochman et al., 1988) uses standard PCR (polymerase chain reaction)- primers oriented in the reverse direction of the usual orientation.
The template for the reverse primers is a restriction fragment that has been selfligated
Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA.
Application
Amplification and identification of flanking sequences such as transposable elements, and the identification of genomic inserts.

46. Long PCR
Extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb).
Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together.
Application- to clone large genes

47. Reverse Transcriptase PCR
Based on the process of reverse transcription, which reverse transcribes RNA into DNA and was initially isolated from retroviruses.
First step of RT-PCR - "first strand reaction“-Synthesis of cDNA using oligo dT primers (37°C) 1 hr.
“Second strand reaction“-Digestion of cDNA:RNA hybrid (RNaseH)-Standard PCR with DNA oligo primers.
Allows the detection of even rare or low copy mRNA sequences by amplifying its complementary DNA.

48. Why real time PCR ? northern blotting ribonuclease protection assay
QUANTITATION OF mRNA
northern blotting
ribonuclease protection assay
in situ hybridization
RT-PCR
most sensitive
can discriminate closely related mRNAs
technically simple
but difficult to get truly quantitative results using conventional PCR
Northern blotting and RPAS are the gold standards, since no amplification is involved.
In situ hybridization is qualitative rather than quantitative.
Techniques such as Northern blotting and ribonuclease protection assays (RPAs) work very well, but require more RNA than is sometimes available. PCR methods are particularly valuable when amounts of RNA are low, since the fact that PCR involves an amplification step means that it is more sensitive. However, traditional PCR is only semi-quantitative at best, in part because of the insensitivity of ethidium bromide (however, there are more sensitive ways to detect the product) and, in part, as we shall discuss later, because of the difficulties of observing the reaction during the truly linear part of the amplification process. Various competitive PCR protocols have been designed to overcome this problem but they tend to be cumbersome.
Real-time PCR has been developed so that more accurate results can be obtained. An additional advantage of real-time PCR is the relative ease and convenience of use compared to some of these older methods (as long as one has access to a suitable real-time PCR machine).

49. Real-Time PCR
Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection

50. Traditional PCR has advanced from detection at the end-point of the reaction to detection while the reaction is occurring (Real-Time).
Real-time PCR uses a fluorescent reporter signal to measure the amount of amplicon as it is generated. This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.

51. Real-time PCR advantages
* amplification can be monitored real-time
* no post-PCR processing of products
(high throughput, low contamination risk)
* ultra-rapid cycling (30 minutes to 2 hours)
* wider dynamic range of up to 1010-fold
* requirement of 1000-fold less RNA than conventional assays
(6 picogram = one diploid genome equivalent)
* detection is capable down to a two-fold change
* confirmation of specific amplification by melting curve analysis
* most specific, sensitive and reproducible
* not much more expensive than conventional PCR
(except equipment cost)

52. Real-time PCR disadvantages
* Not ideal for multiplexing
* setting up requires high technical skill and support
* high equipment cost
* intra- and inter-assay variation
* RNA liability
* DNA contamination (in mRNA analysis)

53. Applications of PCR Detection of pathogens Classification of organisms
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis
Classification of organisms
Genotyping
Molecular archaeology
Mutagenesis
Mutation detection
Sequencing
Cancer research

54. PCR Virtues High sensitivity Can detect and quantify specific events
Higher stability of DNA permits analysis of food samples.
Quantitative and qualitative

55. Thank you
THANK YOU