Polymerase Chain Reaction: Diagnostic Application

1

Polymerase Chain Reaction Diagnostic Application
By Salwa Hassan Teama
Roche
2
Polymerase Chain Reaction Diagnostic Application
By Salwa Hassan Teama M.D. N.C.I. Cairo
University, Egypt
3
Contents

• 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
• http//seqcore.brcf.med.umich.edu/doc/educ/dnapr
  /mbglossary/mbgloss.html

4
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.

5

• The polymerase chain reaction 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. RNA can be
  amplified if converted to cDNA by reverse
  transcriptase.
• Starting materials for gene analysis may be
• Genomic DNA
• RNA
• Nucleic acid from archival material
• Cloned DNA
• PCR products

Croptechnology
Croptechnology

6
Requirements of the reaction

• Template DNA previously isolated and purified
• Two primers to flank the target sequence
• Four normal deoxynucleosides (dNTPs) to
  provide energy and nucleosides for the synthesis
  of DNA
• Buffer system containing magnesium
• DNA polymerase ( thermostable or heat-stable Taq
  polymerase isolated and purified from Thermus
  aquaticus, a bacterium lives in hot springs)

7

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
• Human genomic DNA should be up to 500 ng
• 1-10 ng bacterial DNA
• 0.1-1 ng plasmid DNA

8
Requirements of the reaction

• Primers Oligonucleotide primers are synthesized
  by the DNA synthesizers. They are generally
  synthesized in the range 18-30 nucleotides.
  Typical primers are 18-28 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.

9
Requirements of the reaction

• Buffer system The standard PCR buffer contains
• 1.5 mM MgCL2
• 10 mM Tris HCl (PH 8.4)
• 50 mM KCl
• 100 ? g/ml gelatin or BSA (bovine serum albumin)
• 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
  1mM-5mM, 1.5 mM is optimal in most cases.

10
Requirements of the reaction

• 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.

11
Requirements of the reaction

• 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.

12
Thermal Cycling Profile for Standard PCR

• Initial Denaturation
• Initial heating of the PCR mixture for 2
  minutes at 94- 95C ? 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 snap-back very
  quickly, preventing efficient primer annealing
  and extension or leading to self priming which
  can lead to false positive result.

13

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.
• 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.

14

Primer Anna
Primer annealing
Roche
During PCR, high temperature is used to separate
the DNA molecules into single strands, and
synthetic sequences of single-stranded 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.

15
Roche Molecular Biochemicals PCR Application
Manual
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

16
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.
• 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 ?.

17
Thermal Cycling Profile for Standard PCR
94 C ?
Den.
Ext.
Post- Ext.
72C ?
Ann.
54 C ?
Holding
4 C ?
Hot start time
One cycle repeated 25-35 times
Post-extension time
18

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 (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.

19

PCR Phases Three Phases

• www. AppliedBiosystem.COM Real Time PCR

www.appliedbiosystems.com

20
Plateau Effect

• 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

21
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.

22

Gel Electrophoresis
Well
DNA ladder

23
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.

24

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.

25


Roche Molecular Biochemicals PCR Application
Manual
Reverse transcriptase-PCR (RT-PCR) Roche
Molecular Biochemicals PCR Application Manual



26
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.

27

PCR Methods

• 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.

28
Polymerase Chain Reaction (PCR)

• Advantages of PCR
• Useful non- invasive procedure.
• Simplicity of the procedure.
• Sensitivity of the PCR.
• Disadvantages of PCR
• False positive results (cross contamination).
• False negative results (rare of circulating fetal
  cells).

29

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.

30
The Evolution of PCR to Real-Time
The Evolution of

• www. AppliedBiosystem.COM Real Time PCR

End point detection
www.appliedbiosystems.com
31

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.

32
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 .

33
PCR assays can be performed directly on
genomic DNA samples to detect translocation-specif
ic malignant cells at a sensitivity which is at
least 10,000 fold higher than other methods .
Polymerase Chain Reaction Uses

• t(821) translocation or AML1-ETO fusion gene
• t(1517) translocation or PML-RARA fusion gene
• INV(16) or MYH11-CBFB fusion gene
• t(922) translocation or BCR-ABL fusion gene
  (p210 and p185
• FLT3 Mutations
• BCR-ABL Mutations

34
Polymerase Chain Reaction Uses

• Recurrence of hematological cancers has also
  been evaluated
• 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.
• Monitoring the MRD in leukemia and lymphoma
  patients by assessing PRAME (Preferentially
  expressed antigen of melanoma) in peripheral
  blood samples.

35

Polymerase Chain Reaction Uses

• 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 slow-growing 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.

36
Polymerase Chain Reaction Uses

• The basis for PCR diagnostic applications in
  microbiology is the detection of infectious
  agents and the discrimination of non-pathogenic
  from pathogenic strains (e.g. E.coli) by virtue
  of specific genes.
• PCR primers have also been reported for
  intracellular parasites like T.gondij ,
  P.falciparum and for different strains of
  Trypanosoma,.
• In virology a large number of PCR assays have
  been described for the Human immunodeficiency
  viruses , CMV , HBV ,HSV and others..

37
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 PCR-single-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

38
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.

39
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,..

40
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.

41
References Online Further Reading

• Velasco J. A new view of malignancy New York
  TimesApril 9, 2002..
• Watson JD, Crick FHC. Molecular structure of
  nucleic acids. Nature. 1953171737738. PubMed
• 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. 1953171737738. 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/molekular
  biologie/rt-pcr.jpg