Transcript Slide 1

Polymerase Chain Reaction: Diagnostic Application
By
Salwa Hassan Teama
Roche
Polymerase Chain Reaction: Diagnostic Application
By
Salwa Hassan Teama
M.D. N.C.I. Cairo University, Egypt
Contents
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Standard Polymerase Chain Reaction (PCR)
Requirements of the reaction
Thermal Cycling Profile for Standard PCR
Number of Cycles
PCR Phases: Three phases:
PCR Products
PCR Methods
The Evolution of PCR to Real-Time
Polymerase Chain Reaction: Uses
PCR protocols: http://www.protocolonline.org/prot/Molecular_Biology/PCR/
Molecular Biology Glossary online
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http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.html
Standard Polymerase Chain Reaction (PCR)
Polymerase chain reaction 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 an simple methods for making multiple copies of a DNA
sequence. Developed by researchers at cetus Corporation
(Saiki et al., 1985) ; (Mullis and Faloona. 1987). PCR uses a
thermostable DNA polymerase to produce a 2 fold amplification
of target genetic material with each temperature cycle. The
PCR uses two oligonucleotide primer that are complementary
to nucleic acid sequences flanking the target area , it has
become the most widely used nucleic acid amplification
technology and gold standard for amplification processes in
diagnosis.
The polymerase chain reaction is a test
tube system for DNA replication that
allows a "target" DNA sequence to be
selectively amplified, several millionfold in just a few hours. RNA can be
amplified if converted to cDNA by
reverse transcriptase.
Starting materials for gene analysis may be:
•Genomic DNA
•RNA
Croptechnology
•Nucleic acid from archival material
•Cloned DNA
•PCR products
Croptechnology
Requirements of the reaction
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Template DNA: previously isolated and purified
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Two primers: to flank the target sequence
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Four normal deoxynucleosides (dNTPs) : to provide energy
and nucleosides for the synthesis of DNA
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Buffer system containing magnesium
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DNA polymerase ( thermostable or heat-stable Taq polymerase
isolated and purified from Thermus aquaticus, a bacterium lives
in hot springs)
Requirements of the reaction
Template DNA : Sample preparation by DNA extraction. The
quality of the template influences the outcome of the PCR. If
large amount of RNA in DNA template can chelate Mg+ and
reduce the yield of the PCR. Also impure templates may
contain polymerase inhibitors that decrease the efficiency of the
reaction. The integrity of the template is also important.
Template DNA should be of high molecular weight. To check the
size and quality, run an aliquot on an agarose gel.
 The amount of template in a reaction strongly influences
performance in PCR. The recommended amount of template
for standard PCR is:
The maximum amount of :
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Human genomic DNA should be up to 500 ng
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1-10 ng bacterial DNA
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0.1-1 ng plasmid DNA
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Requirements of the reaction
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Primers: Oligonucleotide primers are synthesized by
the DNA synthesizers. They are generally synthesized
in the range 18-30 nucleotides. Typical primers are 1828 nucleotides in length having 50-60% GC
composition. The calculated Tm for a given primer pair
should be balanced. Primer concentration between
0.1 and 0.6 m are generally optimal. Higher primer
concentration may promote mispriming and
accumulation of non specific product and may
increase the probability of generating a template
independent artifact termed primer-dimer. Lower
primer concentration may be exhausted before the
reaction is completed resulting in lower yields of
desired product.
Requirements of the reaction
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Buffer system: The standard PCR buffer
contains
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1.5 mM MgCL2
10 mM Tris HCl (PH 8.4)
50 mM KCl
100  g/ml gelatin or BSA (bovine serum albumin)
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4.
Mg concentration affects the reaction such that too
little reduces yield and too much increases non
specific amplification. The optimal MgCl2
concentration may vary from approximately 1mM5mM, 1.5 mM is optimal in most cases.
Requirements of the reaction
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dNTP The final concentration of dNTPs should be 50-500  M
(each dNTP).
They are usually included at conc. of 200  M for each
nucleotide. Higher concentration promote misincorporation by
polymerase. Always use balanced solution of all four dNTPs to
minimize polymerase error rate. Imbalanced dNTP mixtures will
reduce Taq DNA Polymerase fidelity. For carry over prevention
a higher concentration of dUTP is usually used in place of
dTTP.
N.B.
If you increase the concentration of dNTP you must increase
Mg+ concentration. Increased in dNTP concentration reduce
free Mg+, thus interfering with polymerase activity and
decrease primer annealing.
Requirements of the reaction
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Taq Polymerase
The most widely characterized polymerase is that from Thermus
aquaticus (Taq), which is a thermophilic bacterium lives in hot
springs and capable of growing at 70 -75 C . The purified
protein (Taq enzyme) has a molecular weight of 94 Kd, and has
an optimum polymerization temperature of 70 – 80 C . The
enzyme loses its activity, but is not denatured, at temperature
above 90 C , and its activity is maintained on return to lower
temprature.
0.5 – 2 units/50  l reaction. Too little will limit the amount of
product, while too much can produce unwanted non specific
products.
Thermal Cycling Profile for Standard PCR
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Initial Denaturation
Initial heating of the PCR mixture for 2 minutes at 9495C  is enough to completely denature complex
genomic DNA so that the primer can anneal to the
template as the reaction mix is cooled. If the template
DNA is only partially denatured, it will tend to snapback very quickly, preventing efficient primer
annealing and extension or leading to self priming
which can lead to false positive result.
Thermal Cycling Profile for Standard PCR
Each cycle includes three successive steps:
 Denaturation: One to several minutes at 94-96 C,
during which the DNA is denatured into single strands.
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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: One to several minutes at 72 C , during
which the polymerase binds and extends a
complementary DNA strand from each primer.
Primer Anna
Primer annealing
Roche
During PCR, high temperature is used to separate the DNA
molecules into single strands, and synthetic sequences of singlestranded DNA (20-30 nucleotides) serve as primers.
Two different primer sequences are used to bracket the target
region to be amplified. One primer is complementary to one DNA
strand at the beginning of the target region; a second primer is
complementary to a sequence on the opposite DNA strand at the
end of the target region.
The primer are arranged so that each primer extension reaction
directs the synthesis of DNA towards the other.
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…………….)
Roche Molecular Biochemicals: PCR Application Manual
Roche Molecular Biochemicals: PCR Application Manual
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Number of Cycles
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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.
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Post extension and holding
Cycling should conclude with a final extension at 72 c  for 5
minute to promote completion of partial extension products and
then holding at 4 c .
Thermal Cycling Profile for Standard PCR
94C 
Den.
Ext.
72C 
54 C 
Ann.
Holding
4C
Hot start time
Post- Ext.
One cycle repeated 25-35 times
Post-extension time
PCR Phases: three phases:
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Exponential: Exact doubling of product is
accumulating at every cycle (assuming 100% reaction
efficiency). The reaction is very specific and precise.
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Linear : The reaction components are being
consumed, the reaction is slowing, and products are
starting to degrade.
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Plateau: (End-Point: Gel detection for traditional
methods): The reaction has stopped, no more
products are being made and if left long enough, the
PCR products will begin to degrade.
PCR Phases: Three Phases
www. AppliedBiosystem.COM Real Time PCR
www.appliedbiosystems.com
Plateau Effect
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Plateau effect is used to describe the attenuation in the
exponential rate of product accumulation that occurs during
the late PCR cycles. Depending on reaction conditions and
thermal cycling one or more of the following may influence
plateau:
Utilization of substrates (dNTP or primers)
Stability of reactants (dNTP or enzyme)
End product inhibition
Competition of reactants by non specific products or primer –
dimer
Incomplete denaturation/ strand separation of product at high
product concentration
PCR Products
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. A gel is made by dissolving
agrose in buffer solution, which is then allowed to set in a gel
tray. The gel tray has combs attached to create wells in the gel,
the samples are prepared and added to the well, and then an
electric current is run through the gel apparatus. The DNA
fragments are separated by charge (e.g. large fragment move
more slowly than small fragments) and the relative sizes of
fragments are determined by comparing to a standard DNA
ladder.
Since PCR amplifications can generate microgram quantities of
product, amplified fragments can be visualized easily following
staining with a chemical stain such as ethidim bromide.
Gel Electrophoresis
DNA ladder
Well
PCR Methods
Reverse transcriptase-PCR (RT-PCR):
PCR may be performed with RNA as a starting material.
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.
Reverse transcriptase-PCR (RT-PCR):
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.
Reverse transcription polymerase chain reaction is
widely used in the diagnosis of genetic diseases and,
quantitatively, in the determination of the abundance of
specific different RNA molecules within a cell or tissue.
Reverse transcriptase-PCR (RT-PCR):
Roche Molecular Biochemicals: PCR Application Manual
Roche Molecular Biochemicals: PCR Application Manual
PCR Methods
Nested-PCR is used to increase the specificity of the
PCR technique; two rounds of PCR are performed
consecutively, using two different pairs of primers. The
known sequence is used to design two pairs of
primers. The second round primers (internal) are
located within the desired amplification product
produced by the first round primers (external). It is
highly unlikely that any region of DNA other than the
intended target will allow sequential amplification with
both sets of primers.
PCR Methods
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Quantitative PCR:
The determination or quantitation of the number of
copies of a given gene achieves accurate
estimation of DNA and RNA targets.
Hot-start PCR - to reduce non-specific
amplification.
Multiplex-PCR
Mutagenesis by PCR.
Inverse PCR
Asymmetric PCR.
In Situ PCR.
Polymerase Chain Reaction (PCR)
Advantages of PCR:
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Useful non- invasive procedure.
Simplicity of the procedure.
Sensitivity of the PCR.
Disadvantages of PCR:
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False positive results (cross contamination).
False negative results (rare of circulating fetal
cells).
The Evolution of PCR to Real-Time
Traditional PCR has advanced from detection at the
end-point of the reaction to detection while the
reaction is occurring (Real-Time).
The real time system reduces the time required for PCR
amplification and analysis from hours to minutes, it is
perfectly suited to:
 Monitor amplification online and in real-time
 Quickly and accurately quantify results
 Analyze melting characteristics of PCR product
 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.
The Evolution of PCR to Real-Time
The Evolution of
www. AppliedBiosystem.COM Real Time PCR
End point detection
www.appliedbiosystems.com
The Evolution of PCR to Real-Time
The recent development
of
real time PCR clearly
demonstrates
many advantages over other
existing
method with:
high accuracy
wide dynamic range
specificity
sensitivity
reduced carry over contamination and rapid
accurate and simultaneous quantification of
multiple samples.
Polymerase Chain Reaction clearly has the potential
to become the routine laboratory method for
diagnosis of a variety of human disorders.
Detection of malignant diseases by PCR
 The detection of leukemia and lymphomas by
the PCR method is currently the highest
developed in cancer research and is already
being used routinely .
Polymerase Chain Reaction: Uses
PCR assays can be performed directly on genomic
DNA samples to detect translocation-specific
malignant cells at a sensitivity which is at least
10,000 fold higher than other methods .
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t(8;21) translocation or AML1-ETO fusion gene
t(15;17) translocation or PML-RARA fusion gene
INV(16) or MYH11-CBFB fusion gene
t(9;22) translocation or BCR-ABL fusion gene (p210 and p185
FLT3 Mutations
BCR-ABL Mutations
Polymerase Chain Reaction: Uses
Recurrence of hematological
cancers has also been evaluated
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To measure the risk of relapse of T lineage acute
lymphoblastic leukemia in children, detection and
quantitation of residual leukemic cells that harbor the
TAL deletion.
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Monitoring the MRD in leukemia and lymphoma
patients by assessing PRAME (Preferentially
expressed antigen of melanoma) in peripheral blood
samples.
Polymerase Chain Reaction: Uses
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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. PCR provides a considerable advantage
over other commonly used methods. This is especially
true for the identification of non-cultivatable or slowgrowing microorganisms such as mycobacteria,
anaerobic bacteria etc. or viruses, where tissue culture
assays and animal models have to be used or which
cannot be cultivated at all.
Polymerase Chain Reaction: Uses
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The basis for PCR diagnostic applications in microbiology is the
detection of infectious agents and the discrimination of nonpathogenic from pathogenic strains (e.g. E.coli) by virtue of
specific genes.
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PCR primers have also been reported for intracellular parasites
like T.gondij , P.falciparum and for different strains of
Trypanosoma,….
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In virology a large number of PCR assays have been described
for the Human immunodeficiency viruses , CMV , HBV ,HSV
and others………..
Polymerase Chain Reaction: Uses
*Major
role in the human genome project.
* Single point mutations can be detected by modified PCR
techniques such as the ligase chain reaction (LCR) and PCRsingle-strand conformational polymorphisms (PCR-SSCP)
analysis.
*Detection of variation and mutation in genes using primers
containing sequences that were not completely complementary
to the template.
* 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).
*Amplification of archival and forensic material
Polymerase Chain Reaction: Uses
* Extending PCR to the amplification of more than one sequence
at a time ( multiplex PCR) made it possible to compare two or
more complex genomes, for instance to detect chromosomal
imbalances.
* Combining in situ hybridization with PCR made possible the
localization of single nucleic acid sequences on one
chromosome within an eukaryotic organism.
* Detection of micro-metastasis in blood, lymph nodes and bone
marrow.
* HLA Typing.
* Analyzing the expression of cytokeratin-18 mRNA in
gastrointestinal carcinoma cell lines.
* DNA analysis for genetic disease diagnosis.
Application of real time PCR in molecular diagnosis
Clinical Microbiology
 Viral load (HIV,HCV,HBV,…)
 Bacterial load (Salmonella, Mycobacterium,..)
 Fungal load( Candida, Cryptococcus, Aspergillus,….)
Food microbiology
 Bacterial load (Listeria, Salmonella, Campylobacter,…)
Clinical Oncology
 Minimal residual disease
 Chromosomal translocations
 Single nucleotide polymorphism (SNPs)
Gene therapy
 Gene transfer estimation
 Biodistribution of vector
Gene expression
 Cytokines, receptors,……..
Conclusion
Polymerase Chain Reaction clearly has the potential to
become the routine laboratory method for diagnosis of
a variety of human disorders. Most clearly, the
detection of infectious agents surpasses current
routine methods.
PCR has very quickly become an essential tool for
improving human health and human life.
References & Online Further Reading
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Velasco J .A new view of malignancy New York TimesApril 9, 2002..
Watson JD, Crick FHC. Molecular structure of nucleic acids .Nature .
.738–171:737;1953PubMed
Osler, W .The Principles and Practice of Medicine .New York: Appleton; 1892 .
Stites DP . Medical immunology. Section II. Page 270
Amr Karim. Workshop on molecular biology and genetic engineering. Faculty of
science . Ain Shams University
WWW.medscape.com
www. pubmedcentral.nih
www.Roche Molecular Biochemicals: PCR Applications Manual
www.Roche Molecular Biochemicals: PCR Techniques
www. AppliedBiosystem.COM Real Time PCR
Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature.
738–171:737;1953. [PubMed]
Osler, W. The Principles and Practice of Medicine. New York: Appleton; 1892
http://en.wikipedia.org/wiki/RT-PCR
http://www.ma.uni-heidelberg.de/inst/ikc/molekularbiologie/rt-pcr.jpg