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
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
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 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
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)
Requirements of the reaction
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
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.
0.1-1 ng plasmid DNA
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 T should be balanced. Primer concentration between 0.1 and 0.6 m for a given primer pair 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:
contains The standard PCR buffer 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 MgCl 5mM, 1.5 mM is optimal in most cases.
2 concentration may vary from approximately 1mM-
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 dTTP.
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 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
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 above 90 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 , and its activity is maintained on return to lower temprature. 0.5 – 2 units/50 products.
l reaction. Too little will limit the amount of product, while too much can produce unwanted non specific
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.
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.
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.
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
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 minute to promote completion of partial extension products and then holding at 4 c .
for 5
94 C
Thermal Cycling Profile for Standard PCR
Den.
72 C 54 C 4 C Ann.
Ext.
Post- Ext.
Holding Hot start time One cycle repeated 25-35 times Post-extension time
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.
PCR Phases: Three Phases
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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
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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 located within the desired amplification product produced by the first round primers highly unlikely that any region of DNA other than the intended target will allow sequential amplification with both sets of primers. (internal) (external).
are It is
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 amplification.
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to reduce non-specific Multiplex-PCR Mutagenesis by PCR .
Inverse PCR Asymmetric PCR.
In Situ PCR .
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).
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
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This kinetic PCR allows for data collection after each cycle of PCR instead of only at the end of the 20 to 40 cycles.
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End point detection
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 .
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
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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.
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.
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 ………..
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
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 Gene therapy Gene transfer estimation Chromosomal translocations Single nucleotide polymorphism (SNPs) 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
Velasco J .A new view of malignancy New York TimesApril 9, 2002..
Watson JD, Crick FHC. Molecular structure of nucleic acids .Nature .
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738 – 171:737 ; 1953 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. 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