Basic intro of PCR

1. POLYMERASE CHAIN
REACTION
PRESENTER : Vigneshwaran V
MODERATOR : Dr.Balasubramaniyan V 1

2. Introduction
Evolution of PCR
Principle
Instrumentation
Reagents
Primer Designing
PCR types
Applications
Summary 2

3. INTRODUCTION
3

4. DNA Replication
• Process of producing two identical copies of DNA from original DNA molecule.
4

5. PCR
• Polymerase chain reaction (PCR) - amplify a Known sequence of DNA to several orders of
magnitude.
• Chain reaction – Small DNA fragment of interest acts as template for the synthesis of new DNA
strand.
• One DNA molecule is used to produce multiple copies exponentially.
• DNA polymerase will add nucleotides to the free 3-OH of this primer according to the normal
base pairing rules.
• PCR typically amplifies DNA fragments of between 0.1 and 10 kilo base pairs.
5

6. 6

7. CELLULAR MACHINERY PCR
MELTING DNA HELICASE HEAT
POLYMERIZING DNA DNA POLYMERASE Taq POLYMERASE
PRIMER PROVIDER PRIMASE PRIMERS IN REACTON MIX
PRIMER RNA DNA
FIDELITY HIGH LESS
In-vivo vs In-vitro Replication of DNA
7

8. Evolution of PCR
1971 – Gobind
Khorona
described
replication of
short fragments
of DNA using
Primers and
polymerases in
vitro.
1976 – Isolation
of DNA
polymerase from
Thermus
aquaticus.
1983 -Kary Mullis
invented PCR.
1985 -Mr.Cycler
was invented.
1986 – Purified
Taq polymerase
first used in PCR.
1988 – Perkin
Elmer introduces
the automated
thermo cycler.
8

9. PRINCIPLE
9

10. Principle
• PCR consists of a series of 20–40 cycles .
• One PCR cycle comprises of 3 steps
• Denaturation
• Annealing
• Extension
• Temperatures used and the length of time applied in each cycle depends on Tm of primers,
DNA polymerase, dNTPs & divalent ion concentration.
10

11. Initialization step
• Usually required for DNA polymerases that are to be activated in Hot-start PCR.
• Heating around the temperature of 94–96 °C for 10 minutes.
• Heating dissociates the inhibitor-DNA polymerase complex.
Step I - Denaturation
• First cycling event
• Heating reaction mixture 94–96 °C for 30 seconds
• DNA melting by disrupting the hydrogen bonds between bases yields single stranded DNA.
11

12. Step II - Annealing
• Reaction temperature is lowered to 50–65°C for 20 – 40 seconds.
• Primer anneals to the complementary bases on flanking region of template DNA.
• Low temperature - primer could bind imperfectly.
• High temperature - primer might not bind
• Annealing temperature 3–5 °C below the Tm of the primers used
12

13. Step III - Extension
• Temperature depends on polymerase used.
• Taq polymerase has its optimum activity temperature at 75–80 °C.
• DNA polymerase synthesizes new DNA strand complementary to the DNA template strand by
adding dNTPs.
• Extension time depends on DNA polymerase used as well as length of the DNA fragment to
amplify.
• At its optimum temperature, the DNA polymerase polymerizes a thousand bases per minute.
13

14. 5’
5’
3’
3’
d.NTPs
Thermal Stable
DNA Polymerase
Primers
Denaturation
Annealing
Add to Reaction Tube
14

15. Extension
5’ 3’
5’3’
Extension Continued
5’ 3’
5’3’
Taq
Taq
3’
5’3’
Taq
Taq
Repeat
15

16. PCR Steps
Cycling: Repeat
steps 1 through 3 (20 - 40 times)
Extend primers,
yielding new double-stranded DNA
Anneal
primers to single-stranded DNA
Denature
double-stranded DNA
16

17. CYCLE NUMBER DNA copy number
0 1
1 2
2 4
3 8
4 16
5 32
6 64
7 128
8 256
9 512
10 1,024
11 2,048
12 4,096
13 8,192
14 16,384
15 32,768
16 65,536
17 131,072
18 262,144
19 524,288
20 1,048,576
21 2,097,152
22 4,194,304
23 8,388,608
24 16,777,216
25 33,554,432
26 67,108,864
27 134,217,728
28 268,435,456
29 536,870,912
30 1,073,741,824
31 1,400,000,000
32 1,500,000,000
33 1,550,000,000
34 1,580,000,000
Copies of DNA=2N
0.0E+00
2.0E+08
4.0E+08
6.0E+08
8.0E+08
1.0E+09
1.2E+09
1.4E+09
1.6E+09
1.8E+09
0 5 10 15 20 25 30 35
PCR cycle
DNAcopynumber
17

18. REAGENTS &
INSTRUMENTATION
18

19. PCR Requirements:
• DNA template containing desired segment
• Primers
• DNA polymerase
• Deoxynucleoside triphosphates dNTPs.
• Buffers
• Bivalent cations – Mg2+ , Mn2+
• Monovalent cation - K+
• Additives
19

20. Thermal cycler
• Earliest thermal cyclers were designed for use with the Klenow
fragment of DNA polymerase I.
• Peltier element – Modern PCR machines.
• Lid temperature - 105oC.
• Thermal blocks – 48/96 wells.
20

21. 21

22. PRIMER DESIGNING
22

23. Primer
• Primer is an oligonucleotide sequence – 18-26 bp in length
provides free 3’OH for the attachment of nucleotide bases
by Polymerase.
• Primers need to match the beginning and the end of the
DNA fragment to be amplified.
• In PCR, both the strands will be amplified. So, one primer
each for both the strands must be designed.
• Forward primer - beginning of gene of interest.
• Reverse primer beginning of complementary strand (in the
5' end). 23

24. Primer length
Primer melting temperature
• Optimal length of PCR primers is 18-26 bp.
• Long enough for adequate specificity and short enough for primers to bind easily to the
template.
• Temperature at which one half of the DNA duplex will dissociate to become single
stranded.
• Primers with melting temperatures in the range of 52-58oC produce the best results.
• GC content of the sequence gives a fair indication of the primer Tm
24

25. Annealing Temperature (Ta)
• Annealing temperature (Ta) relies directly on length and composition of the primers.
• Ta must be set 5oC below the Tm of your primers.
• High Ta - insufficient primer-template hybridization
• low Ta - non-specific binding
GC content
• GC content of the primer should be 40-60%
25

26. • Presence of G or C bases within the last five bases of primers (GC clamp) promote specific
binding.
• More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
GC clamp
Primer Secondary Structures
• Intermolecular or intramolecular interactions creates primer secondary structures leads to
poor or no yield of the product.
• Affects primer template annealing and thus the amplification.
26

27. Repeats
• Di-nucleotide occurring many times consecutively.
• Maximum number of di-nucleotide repeats acceptable in an oligo is 4 di-nucleotides.
• Primers with long runs of a single base should be avoided.
• Maximum number of runs accepted is 4bp.
Runs
Avoid cross homology
• To improve specificity of the primers it is necessary to avoid regions of homology.
• Primers designed for a sequence must not amplify other genes in the mixture.
• Primers are designed and then BLASTed to test the specificity
27

28. DNA POLYMERASES
28

29. Taq polymerase
• Thermophilic bacterium - Thermus aquaticus
• Optimum temperature - 70–80°C
• Half-life - 40 minutes at 95°C
• Lacks 3’-5’ proofreading activity.
• 1 error in every 104 nucleotides incorporated.
pfu DNA Polymerase
• Isolated from Pyrococcus furiosus.
• 3’-5’ & 5’-3’ exonuclease activity
• Fidelity of enzyme is 12 fold higher.
• Half life at 95°C – 2 hours
29

30. Pwo DNA Polymerase
• Isolated from Pyrococcus woesei
• Possess 3’-5’ exonuclease proof reading activity.
• Half life – 2 hours & 5 minutes at 100°C
• Fidelity of enzyme is 10 fold higher than Taq polymerase.
• Processevity rate is same as Taq polymerase.
Accuzyme DNA Polymerase
• 47 folds higher fidelity compared to Taq polymerase.
• Polymerase with highest fidelity
• Possess 5’-3’ polymerase activity & 3’-5’ proof reading activity
30

31. KOD Hifi DNA Polymerase
• Isolated from Thermus thermophilus.
• Does not possess a proofreading activity.
• Possess reverse transcriptase (RT) activity.
• RT activity is dependent upon presence of 1–2 mM manganese ions while the DNA
polymerase functions best in the presence of magnesium ions.
• Optimum temperature is 70°C
Tth DNA polymerase
• Isolated from Thermococcus kodakaraensis.
• High processivity and fidelity.
• Elongation rate is 5 times faster and processivity 10–15 times higher than for Pfu DNA
polymerase
31

32. M-MLV Reverse Transcriptase
• Purified from E. coli expressing the pol gene of M-MLV
• Half-life of 220 minutes at 50°C
• SuperScript® III RT is genetically engineered by the introduction of point mutations that
increase half-life, reduce RNase activity, and increase thermal stability.
Superscript III Reverse Transcriptase
• Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) – RNA dependent DNA
polymerase.
• Synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA
hybrid.
• Lacks DNA endonuclease activity
32

33. 33

34. PCR ADDITIVES
34

35. • BSA (usually at 0.1 to 0.8 µg/µL final concentration)
• Stabilize Taq polymerase & overcome PCR inhibitors
• DMSO (usually at 2-5% v/v, inhibitory at ≤ 10% v/v)
• Denaturant – Prevents secondary structure formation in GC rich template & primers.
• Glycerol (usually at 5-10% v/v)
• Increases apparent concentration of primer/template mix, and often increases PCR efficiency at high
temperatures.
• Stringency enhancers (Formamide, Betaine, TMAC)
• Enhances yield and reduces non-specific priming
• Non-ionic detergents (Triton X, Tween 20 or Nonidet P-40) (0.1–1%)
• Stabilize Taq polymerase & suppress formation of 2º structure
35

36. BUFFERS
36

37. KCl
• Promotes primer-template annealing.
• High concentrations – Stabilizes primers annealed to non target sites.
Mg2+
• Important component of PCR.
• Taq DNA polymerase dependent on Mg2+
• Taq DNA polymerase shows its highest activity around 1.2–1.3 mM free
Mg2+.
• Magnesium concentration can also affect the fidelity (error rate) of DNA
polymerases.
37

38. TYPES OF PCR
TYPES OF PCR
38

39. Reverse Transcriptase -PCR
• Detect gene expression through the synthesis of complementary DNA (cDNA) transcripts from RNA.
• RNA template converted into a complementary DNA (cDNA) using a reverse transcriptase.
• Primers – Oligo dt, random primers & gene specific primers.
• One step & Two step RT-PCR.
• Amplified DNA fragments that are produced
can by analyzed by agarose gel electrophoresis.
• Amount of amplified fragment produced
proportional to the amount of target mRNA in
the original RNA sample.
39

40. Real Time PCR or (q-PCR)
• In RT-PCR, process of amplification of DNA is monitored in real time.
• PCR with an added probe or dye to generate a fluorescent signal from the product.
• Detection of signal in real time allows quantification of starting material.
• Performed in specialized thermal cyclers with fluorescent detection systems.
• PCR signal is observed as an exponential curve with a lag phase, a log phase, a linear phase,
and a stationary phase.
40

41. Detection Systems
• Non-Specific Detection
• SyBr green, BEBO, BOXTO, Eva Green.
• Specific Detection
• Taqman probe, Molecular Beacon, Light-up probes & Hybridization Probes.
• Primer Based Detection
• Scorpion primers, Qzyme & Lux primers.
41

42. SyBr green
Extension
5’ 3’
5’3’
Apply Excitation
Wavelength
5’ 3’
5’3’
Taq
Taq
3’
5’3’
Taq
Taq
Repeat
ID ID
ID IDID
ID ID ID
ID ID
l l l
l
l
ID ID
ID ID
• Binds to minor groove (dsDNA).
• Non-specific
• Increased quantity of dsDNA = Increased
binding of SYBR green = Increased
fluorescence.
42

43. Taqman Probe
5’ 3’
RQ
5’ 3’
Taq
Q
R
5’ 3’
Q
Taq
R
5’
5’ 3’
Taq
R
5’
5’ 3’
Taq
R
5’
l
Extension
Hydrolysis
Signal
• Relies on the 5-3´ exonuclease activity
of Taq polymerase.
• Fluorophore (R) - 5’-end
Quencher (Q) - 3’-end.
• Quencher molecule quenches
the fluorescence emitted by the fluorophore
when they are close to each other.
• Degradation of the probe - releases fluorophore -
relieving quenching effect - allowing fluorescence.
43

44. Molecular Beacon Assay
• Hairpin shaped molecules with an internally
quenched fluorophore.
• 25 nucleotides long - middle 15 nucleotides are
complementary to target nucleotides & five
nucleotides at each terminus are complementary
to each other.
• 5’ – Fluorophore & 3’ – Quencher.
• Nucleic acid to be detected is complementary to
the strand in the loop, event of
hybridization occurs & relieves quenching effect
on fluorophore.
44

45. 45
www.biorad.com
2a. excitation
filters
2b. emission
filters
1. halogen
tungsten lamp
4. sample plate
3. intensifier
5. ccd
detector
350,000
pixels
qPCR Optical Systems

46. 46
Amplification Curve
• Amplification plot – 3 phases –
• Initiation phase
• Exponential phase
• Plateau phase
• Ct (cycle threshold) - number of cycles required
for the fluorescent signal to cross the threshold
(ie exceeds background level).
• Small number of template at initial cycles –
(Increased Ct number) more amplification cycles
required to attain Ct .

47. Data Analysis
• Levels of expressed genes may be measured by absolute
or relative quantitative real-time PCR
• Absolute Quantification
• Quantitate unknowns based on a known quantity.
• Samples of known DNA concentration were amplified
along with samples of unknown DNA concentration,
Ct values were obtained.
• Plot a graph – Ct (Y-axis); DNA concentration (X-axis) –
concentration of DNA in unknown sample obtained by
comparing unknown to the standard curve.
47
• Relative Quantification
• Based on the expression levels of a target gene versus an housekeeping gene.
• Determines the changes in steady-state mRNA levels of a gene across multiple samples
and expresses it relative to the levels of an internal control RNA

48. • Formation of large oligo nucleotides of DNA from
short segments
• Each oligonucleotide is designed to be either part of
the top or bottom strand of the target sequence.
• Oligonucleotides anneal to complementary
fragments and then are filled by polymerase.
• Each cycle thus increases the length of various
fragments randomly depending on which
oligonucleotides find each other.
• Production of synthetic genes and even entire
synthetic genomes.
Assembly PCR
48

49. Asymmetric PCR
• Amplifies one strand of the target DNA
• Used in sequencing
and hybridization probing - amplification of
only one of the two complementary
strands is required.
• PCR is carried out with a great excess of
primer for the strand targeted for
amplification
49

50. Colony PCR
• Screening of bacteria (E.coli) or yeast clones for correct
ligation or Plasmid products.
• Individual transformants can either be lysed in water with a
short heating step or added directly to the PCR reaction and
lysed during the initial heating step.
• Initial heating step causes the release of the plasmid DNA
from the cell, so it can serve as template for the
amplification reaction.
• Primers designed to specifically target the insert DNA can be
used to determine if the construct contains the DNA
fragment of interest and also insert orientation. 50

51. Hot-start PCR
• Hot-start PCR prevents non-specific
amplification & primer-dimer formation.
• Performed manually by heating the reaction
components to the melting temperature (e.g.,
95˚C)
• Antibodies, Affibodies, Chemical modifications
& Aptamer inhibits polymerase activity at
ambient temperature only dissociate after a
high temperature activation step (95˚C).
51

52. 52

53. Inverse PCR
• Method used to allow PCR when only one internal
sequence is known.
• This is especially useful in identifying flanking
sequences to various genomic inserts.
• This involves a series of DNA digestions and self
ligation, resulting in known sequences at either
end of the unknown sequence.
53

54. • Prevents non-specific binding of primer and its
amplification.
• Two sets of primers, used in two successive runs of
polymerase chain reaction.
• First primer binds to the region far away from Target
sequence and product is formed.
• Products are then used in a second PCR reaction with
second set of primers whose 3’ end complementary to
Target sequence.
• second PCR has little contamination from unwanted
products of primer dimers, hairpins, and alternative
primer target sequences.
Nested PCR
54

55. Touch down PCR
• Reduce nonspecific background amplification -lowering the
annealing temperature as PCR cycling progresses.
• Annealing temperature at the initial cycles is usually a few
degrees (3-5˚C) above the Tm of the primers used, later
cycles, it is a few degrees (3-5˚C) below the primer Tm.
• Higher temperatures give greater specificity for primer
binding, and the lower temperatures permit more efficient
amplification from the specific products formed during the
initial cycles.
55

56. APPLICATIONS
56

57. Applications of PCR
Molecular Identification Sequencing Genetic Engineering
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies
57

58. Huntington’s Disease (HD)
• HD is caused by a mutation in the Huntingtin (HD) gene
• In non-HD individuals, the HD gene has a pattern called trinucleotide repeats with
“CAG” occurring in repetition less than 30 times.
• IN HD individuals, the “CAG” trinucleotide repeat occurs more that 36 times in the
HD gene
• PCR can be performed on an individual’s DNA to determine whether the individual has
HD.
• The DNA is amplified via PCR and sequenced (a technique by which the exact
nucleotide sequence is determined) and the number of trinucleotide repeats is
then counted.
58

59. Cystic Fibrosis (CF)
• CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator
(CTFR) gene.
• In non-CF individuals, the CTFR gene codes for a protein that is a chloride ion channel
and is involved in the production of sweat, digestive juices and mucus.
• In CF individuals, mutations in the CTFR gene lead to thick mucous secretions in the
lungs and subsequent persistent bacterial infections.
• The presence of CTFR mutations in a individual can be detected by performing PCR and
sequencing on that individual’s DNA.
59

60. Human Immunodeficiency Virus (HIV)
• HIV tests rely on PCR with primers that will only amplify a section of the viral DNA
found in an infected individual’s bodily fluids.
Therefore if there is a PCR product, the person is likely to be HIV positive.
If there is no PCR product the person is likely to be HIV negative.
60

61. 61

62. Summary
• Polymerase chain reaction - (DNA PHOTOCOPIER) amplify Known sequence of DNA to several
orders of magnitude.
• PCR consists of a series of 20–40 cycles consisting of 3 steps – Denaturation, Annealing &
Extension.
• Primer provides free 3’OH for the attachment of nucleotide bases by Polymerase.
• Heat resistant DNA polymerase – Taq Polymerase, Pfu Polymerase.
• PCR additives & Buffers increases the binding affinity of primers to the template strand and
also increases polymerase activity.
• Reverse transcriptase PCR – converts RNA to cDNA – One step & Two step 62

63. • Real time PCR – Process of DNA amplification is monitored in real time – used to
quantitate DNA in the samples.
• Assymetric PCR – Amplifies one strand of target DNA.
• Hot start PCR - prevents non-specific amplification & primer-dimer formation by
inactivating the activity of Polymerase.
• Nested PCR – Prevents non-specific binding of primer and its amplification by using
two sets of primers.
63

64. 64
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