1. 2013:Polymerase Chain Reaction
Shakira Sulehri

2. Contents
 Standard Polymerase Chain Reaction (PCR)
 PCR Reaction Components
 Thermal Cycling Profile for Standard PCR
 PCR Phases
 PCR Products
 Types of PCR
 Polymerase Chain Reaction: Uses

3. Polymerase chain reaction
Polymerase chain reaction was invented by Kary Mullis and his
colleagues in the 1983. Nobel prize 1993. It has become the most
widely used nucleic acid amplification technology and gold standard
for amplification processes in diagnosis and research.
PCR is a technique for in vitro amplification of specific DNA
sequences via the temperature mediated DNA polymerase enzyme by
simultaneous primer extension of complementary strands of DNA.
PCR is a test tube system for DNA replication that allows a "target"
DNA sequence to be selectively amplified, several million-fold in just a
few hours.

4. PCR Reaction Components
 Template: previously isolated and purified.
 Two primers: to flank the target sequence.
 Four deoxynucleosides triphosphate (dNTPs): to
provide energy and nucleosides for the synthesis of
DNA.
 Buffer system containing magnesium.
 DNA polymerase

5. PCR Reaction Components
Template: The recommended amount of template for standard PCR is:
 The maximum amount human genomic DNA should be up to 500 ng.
 1-10 ng bacterial DNA.
 0.1-1 ng plasmid DNA.
Primers: Primer concentration between 0.1 and 0.6 µM are generally optimal.
Higher primer concentrations may promote mispriming and accumulation of
non specific product. Lower primer concentrations may be exhausted before
the reaction is completed, resulting in lower yield of desired product.
Deoxynucleosides Triphosphate (dNTPs) Concentration: Balanced solution of all
four dNTPs minimize polymerase error rate. Imbalanced dNTP mixture will
reduce Taq DNA polymerase fidelity.The final concentration of dNTPs should be
50-500 µ M (each dNTP). They are usually included at conc. of 200 µ M for each.
Buffer system containing magnesium: Providing a suitable chemical environment
for optimum activity and stability of the DNA polymerase. Generally, the Ph of
the reaction buffer is (Ph 8.3 – 9.0) will give the optimal results.
Mg+ Concentration,The optimal MgCl2 concentration may vary from approximately
1mM-5mM, 1.5 mM is optimal in most cases.
DNA Polymerase: The recommended amount is 0.5 – 2.5 units/50 µ l reaction. Too
little will limit the amount of product, while too much can produce unwanted
non specific products and decreased specificity.

6. DNA Polymerase:
 The most widely characterized polymerase is that
from Thermus aquaticus (Taq DNA Polymerase),
which is a thermophilic bacterium lives in hot
springs and capable of growing at 70 -75 C °.
 The purified protein (Taq enzyme) consist of a single
polypeptide chain has a molecular weight of 95 Kd,
and has an optimum polymerization temperature of
70 – 80 C °.

7.  Initial Denaturation:
This step consists of heating the reaction to a temperature of 94-95°C
which is held for 1-9 minutes. Initial heating of the PCR mixture for 2
minutes at 94- 95C °is enough to completely denature complex
genomic DNA.
 Each cycle includes three successive steps: Each cycle
takes as little as few minutes and it usually takes fewer than 20 cycles
to produce as much amplified DNA as one needs.
Denaturation: One to several minutes at 94-96 C°, during which the DNA
is denatured into single strands.
Annealing: One to several minutes at 50-65 C °, during which the primers
hybridize or "anneal" (by way of hydrogen bonds) to their
complementary sequences on either side of the target sequence; and
Extention: For fragments up to 3 kb primer extension is normally carried
out at 72 C °, during which the polymerase binds and extends a
complementary DNA strand from each primer and add approximately
60 bases per second at 72C °.
 Post extension and holding:
Cycling should conclude with a final extension at 72 C ° for 5 -15
minute to promote completion of partial extension products and then
holding at 4 c °.
Thermal Cycling Profile for Standard PCR

8. DenaturationDenaturation
AnnealingAnnealing
ExtentionExtention

9.  End of the 1st PCR Cycle results in two Copies of target sequence. The copies of both
strands then serve as templates for the next round of synthesis.
 As amplification proceeds , the DNA sequence between primers doubles after each cycles
(The amplification of the target sequence proceeding in an exponential fashion ( 1 2
4 8 16…………….) up to million of times the starting amount until enough is present
to be seen by gel electrophoresis.
Number of Cycles:
 The number of cycles required for optimum amplification varies depending on the amount
of the starting material.
 In optimal reaction, less than 10 template molecules can be amplified in less than 40
cycles to a product that is easily detectable on a gel stained with ethidium bromide.
 Most PCR should , therefore, include only 25 – 35 cycles.
 As cycle increases, nonspecific products can accumulate.
 After 20- 40 cycles of heating and cooling build up over a million copies of original DNA
molecules.

11. PCR Phases
Three phases:
 Exponential: Exact doubling of product is accumulating at every cycle
(assuming 100% reaction efficiency). The reaction is very specific and precise.
 Linear: The reaction components are being consumed, the reaction is slowing,
and products are starting to degrade.
 Plateau: The reaction has stopped, no more products are being made and if left
long enough, the PCR products will begin to degrade.

12. Gel Electrophoresis
Following amplification, the PCR products are usually loaded
into wells of an agarose gel and electrophoresed.
 Gel electrophoresis is a method used to separate or purify
samples of DNA , RNA , or protein using an electric current
applied to a gel matrix (porous sponge like matrix).
 Electrophoretic "gels" are composed of either agarose or
polyacrylamide. These two substrates differ in resolving power,
and also in the difficulty of setting them up .
 Agarose gels are used much more commonly except for small
fragments of DNA. Polyacrylamide gels are also widely used for
electrophoresis of proteins.

13. Agarose Gel Electrophoresis:
 It is a method used in biochemistry and molecular biology to separate DNA, or
RNA molecules based upon charge, size and shape.
 A gel is made by dissolving agarose powder in boiling buffer solution, the
solution is then cooled to approximately 50C ° and poured where it solidifies.
The gel is submerged in a buffer filled chamber which contain electrodes.
 The gel tray has combs attached to create wells in the gel, the samples are
prepared and added to the well by mixing them with solutions containing
glycerol or sucrose, and then an electric current is run through the gel
apparatus.
 The DNA fragments are separated by charge and the relative sizes of fragments
are determined by comparing to a standard DNA ladder.
 Factors, such as charge, size and shape, together with buffer conditions, gel
concentration and voltage, affect the mobility of molecules in gels.
 Since PCR amplifications can generate microgram quantities of product,
amplified fragments can be visualized easily following staining with a chemical
stain such as ethidium bromide.

14. Advantages of PCR:
 Simplicity of the procedure.
 Sensitivity of the PCR.
Disadvantages of PCR:
 False positive results (cross contamination).
 False negative results (e.g. rare of circulating fetal
cells

15. Types of PCR
 Reverse transcriptase-PCR.
 Nested-PCR.
 Hot-start PCR.
 Quantitative PCR.
 Multiplex-PCR.
 Mutagenesis by PCR.
 Allele specific PCR.
 Inverse PCR.
 Asymmetric PCR.
 In Situ PCR.
 ….

16. Reverse Transcriptase-PCR
 RT-PCR, one of the most sensitive
methods for the detection and
analysis of rare mRNA transcripts
or other RNA present in low
abundance.
 RNA cannot serve as a template for
PCR, so it must be first transcribed
into cDNA with reverse
transcriptase from Moloney murine
leukemia virus or Avian
myeloblastosis virus, and the cDNA
copy is then amplified.
 The technique is usually initiated by
mixing short (12-18 base) polymers
of thymidine (oligo dT) with
messenger RNA such that they
anneal to the RNA's polyadenylate
tail. Reverse transcriptase is then
added and uses the oligo dT as a
primer to synthesize so-called first-
strand cDNA.
Roche Molecular Biochemicals: PCR Application Manual. RT-PCRRoche Molecular Biochemicals: PCR Application Manual. RT-PCR

17. Nested PCR
 Nested PCR is a variation of the polymerase chain reaction (PCR), in
that two pairs (instead of one pair) of PCR primers are used to amplify a
fragment.
 The first pair of PCR primers amplify a fragment similar to a standard
PCR. However, a second pair of primers called nested primers bind
inside the first PCR product fragment to allow amplification of a second
PCR product which is shorter than the first one.
 Nested PCR is a very specific PCR amplification.

18. Hot Start PCR
 Hot Start PCR significantly improve specificity, sensitivity and
yield of PCR
 Some components essential for polymerase activity is separated
from the reaction mixture until the temperature in the tubes has
exceeded the optimal primer annealing temperature usually 55-65
C ˚.
 The technique may be performed manually by heating the reaction
components to the melting temperature (e.g., 95˚C) before adding
the polymerase. Specialized enzyme systems have been
developed that inhibit the polymerase's activity at ambient
temperature, either by the binding of an antibody or by the
presence of covalently bound inhibitors that only dissociate after
a high-temperature activation step.

19. Real-Time PCR
QUANTITATION

20. Real-Time PCR
PCR was traditionally limited to end-point analysis using agarose
gels
Limitations of end-point PCR:
Poor precision
Low sensitivity
Short dynamic range
Low resolution
Size-based discrimination
Ethidium bromide for staining does not allow for
accurate quantitation
Requires post-PCR processing
Real-time PCR instruments and chemistry
allow for the detection and quantitation of
amplification throughout the reaction

21. End Point
Standard
PCR
Real-Time PCR
Plateau
phase
Linear
phase
Exponential
phase
Area of detection for
real-time PCR

22. QUANTITATION
Theoretically there is a quantitative relationship between the amount of starting sample and
the amount of PCR product at any given sample.
Real-time PCR detects the accumulation of amplicon during the reaction. The data is then
measured at the exponential phase of the PCR reaction rather than end-point plateau. The
exponential phase is the optimal point for analyzing data.
CT
Cycle threshold is
related to the initial
target copy number
Real-Time PCR

23. Applications:
Viral quantitation
Quantitation of gene expression
Microarray verification
Drug therapy efficacy
Pathogen detection
Genotyping
Advantages of real-time vs. end-point PCR:
Collects data in the exponential growth phase (vs end-point plateau(
Increase in fluorescent signal is proportional to number of amplicons
generated
Increased dynamic range of detection
Does not require post-PCR processing
Increased sensitivity (detection down to 2-fold change(
Real-Time PCR

24. Detection Assays: SYBR Green Dye
SYBR Green I binds to double-
stranded DNA. The resulting
DNA-
dye-complex absorbs blue light
(max = 498 nm( and emits green
light, (max = 522 nm(
As DNA is amplified SYBR
fluorescence increases
proportionally
Real-Time PCR
Non-specific dye used to detect the
presence or absence of an
amplicon

25. Dual fluorophore-labeled oligonucleotide
probe: e.g. TaqMan
Detection Assays: Sequence-specific
probes
Real-Time PCR

26. Polymerase Chain Reaction: Uses
The polymerase chain reaction (PCR) is a technique widely
used in:
Molecular biology,
Microbiology,
Genetics,
Diagnostics clinical laboratories,
Forensic science,
Environmental science,
Hereditary studies,
Paternity testing, and
Many other
applications………………………………………

27. Diagnosis of a variety of human disorders:
Infectious agents:
 One area where the PCR technique will undoubtedly become a routine method, is the
detection of infectious agents, such as pathogenic bacteria, viruses or protozoa.
Cancer:
 Detection of malignant diseases by PCR.
 Recurrence of hematological cancers has also been evaluated.
 Detection of micro-metastasis in blood, lymph nodes and bone marrow.
Diagnosis of Genetic Diseases:
 Single point mutations can be detected by modified PCR techniques .
 Detection of variation and mutation in genes using primers containing sequences that
were not completely complementary to the template.
Prenatal Sexing and prenatal diagnosis of diseases:
 Prenatal sexing is often required in families with inherited sex linked diseases. In these
cases chorionic villus samples are ideal material for fetal sexing in the first trimester of
pregnancy.
 Prenatal Diagnosis of diseases
e.g. Prenatal diagnosis of many of the inborn errors of metabolism is possible by DNA
markers.

28.  Mapping of entire genomes .
 Identify the level of expression of genes in extremely small samples of
material, e.g. tissues or cells from the body by reverse transcription-
PCR (RT-PCR).
 Multiplex PCR , made it possible to compare two or more complex
genomes, for instance to detect chromosomal imbalances
 Amplification of forensic material.
• PCR is used in research laboratories in DNA cloning procedures,
Southern blotting, DNA sequencing, recombinant DNA technology.

29. Thank you