Introduction, Evolution of PCR, Principle, Instrumentation, Reagents, Primer Designing, DNA polymerase, Components of PCR, PCR Types, Applications

1. POLYMERASE CHAIN
REACTION
Advanced Pharmaceutical Analysis
Presented by-
Mehul H Jain
M. Pharmacy I-I
Pharmaceutical Analysis

2. CONTENTS
1. Introduction
2. Evolution of PCR
3. Principle
4. Instrumentation
5. Reagents
6. Primer Designing
7. DNA Polymerases
8. Components of PCR
9. PCR types
10. Application

3. INTRODUCTION

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

5. PCR
 PCR, polymerase chain reaction, is an in-vitro technique for amplification of a
region of DNA whose sequence is known or which lies between two regions of
known sequence.
 Before PCR, DNA of interest could only be amplified by over-expression in
cells and this with limited yield.
 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.

6. EXPONENTIAL AMPLIFICATION

7. EVOLUTION OF PCR

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

9. PRINCIPLE

10.  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.
Initialization Step:
 In this step the reaction is heated to 94-96°C for 30 seconds to several minutes. This
step is usually only done in the very beginning of the PCR reaction.
 This step is important for activating hot-start polymerases, if we use such a
polymerase and to denature your template DNA.
 If the template GC content is high we may need to perform an extra-long
initialization step.
 Heating dissociates the inhibitor - DNA polymerase complex.

11. 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.
Step – II: Annealing
 Reaction temperature is lowered to 50 - 65°C for 20 - 40 seconds.
 The temperature in this step needs to be low enough that the denatured primers can
form Watson-Crick base pairs with the template DNA. But high enough that only the
most stable (perfectly paired) double-stranded DNA structures can form.
 Usually this perfect annealing temperature is a few degrees lower than the melting
temperature of the primer pair.
 During this step the polymerase will binds to the primer/template DNA complex.
Although the polymerase will not start reading until the temperature is raised in the
next step.

12. 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.

15. PCR STEPS

16. REAGENTS &
INSTRUMENTATION

17. PCR REQUIREMENTS
 DNA template containing desired segment
 Primers
 DNA polymerase
 Deoxy nucleoside triphosphates dNTPs.
 Buffers
 Bivalent cations – Mg2+ , Mn2+
 Monovalent cation - K+
 Additives

18. 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.
The thermal cycler (also known as a thermocycler, PCR
machine or DNA amplifier) is a laboratory apparatus most
commonly used to amplify segments of DNA via the polymerase
chain reaction (PCR). Thermal cyclers may also be used in
laboratories to facilitate other temperature-sensitive reactions,
including restriction enzyme digestion or rapid diagnostics. The
device has a thermal block with holes where tubes holding the
reaction mixtures can be inserted. The cycler then raises and
lowers the temperature of the block in discrete, pre-programmed
steps.

19. With one cycle, a single
segment of double-stranded DNA
template is amplified into two
separate pieces of double-stranded
DNA. These two pieces are then
available for amplification in the
next cycle. As the cycles are
repeated, more and more copies
are generated and the number of
copies of the template is increased
exponentially.

20. PRIMER DESIGNING

21. 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).

22. Primer Length:
 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.
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.
Primer Melting Temperature:
 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.

23. 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%.
GC Clamp:
 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.

24. DNA POLYMERASES

25. 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.

26. COMPONENTS OF PCR

27. The PCR reaction requires the following components:
 DNA Template: The double stranded DNA (dsDNA) of interest, separated from the
sample.
 DNA Polymerase: Usually a thermostable Taq polymerase that does not rapidly
denature at high temperatures (98°), and can function at a temperature optimum of
about 70°C.
 Oligonucleotide primers: Short pieces of single stranded DNA (often 20 - 30 base
pairs) which are complementary to the 3’ ends of the sense and anti- sense strands of
the target sequence.
 Deoxynucleotide triphosphates: Single units of the bases A, T, G, and C (dATP,
dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks
for DNA synthesis.
 Buffer system: Includes magnesium and potassium to provide the optimal
conditions for DNA denaturation and renaturation; also important for polymerase
activity, stability and fidelity.

28. TYPES OF PCR

29. 1. 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.

30. 2. 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.

31. 3. Assembly PCR:
 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.

32. 4. 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.

33. 5. 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.
6. Hot-start PCR
7. Inverse PCR
8. Nested PCR
9. Touch down PCR

34. APPLICATIONS

36.  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.
 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.

37. THANK YOU