Polymerase Chain Reaction Diagnostic Application By Salwa Hassan

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 n n n 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 millionfold in just a few hours. RNA can be amplified if converted to c. DNA 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 n Template DNA: previously isolated and purified n Two primers: to flank the target sequence n Four normal deoxynucleosides (d. NTPs) : to provide energy and nucleosides for the synthesis of DNA n Buffer system containing magnesium n 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. n 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 : n Human genomic DNA should be up to 500 ng n 1 -10 ng bacterial DNA n 0. 1 -1 ng plasmid DNA n

Requirements of the reaction n 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 n 1. 2. 3. 4. Buffer system: The standard PCR buffer contains 1. 5 m. M Mg. CL 2 10 m. M Tris HCl (PH 8. 4) 50 m. M 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 Mg. Cl 2 concentration may vary from approximately 1 m. M 5 m. M, 1. 5 m. M is optimal in most cases.

Requirements of the reaction n d. NTP The final concentration of d. NTPs should be 50 -500 M (each d. NTP). 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 d. NTPs to minimize polymerase error rate. Imbalanced d. NTP mixtures will reduce Taq DNA Polymerase fidelity. For carry over prevention a higher concentration of d. UTP is usually used in place of d. TTP. N. B. If you increase the concentration of d. NTP you must increase Mg+ concentration. Increased in d. NTP concentration reduce free Mg+, thus interfering with polymerase activity and decrease primer annealing.

Requirements of the reaction n 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 n Initial Denaturation Initial heating of the PCR mixture for 2 minutes at 9495 C 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: n Denaturation: One to several minutes at 94 -96 C , during which the DNA is denatured into single strands. n 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 n 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 n

Number of Cycles n 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. n 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 94 C Den. Ext. 72 C 54 C Ann. Holding 4 C Hot start time Post- Ext. One cycle repeated 25 -35 times Post-extension time

PCR Phases: three phases: n Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. n Linear : The reaction components are being consumed, the reaction is slowing, and products are starting to degrade. n 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. Applied. Biosystem. COM Real Time PCR www. appliedbiosystems. com

Plateau Effect n 1. 2. 3. 4. 5. 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 (d. NTP or primers) Stability of reactants (d. NTP 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 m. RNA transcripts or other RNA present in low abundance. RNA cannot serve as a template for PCR, so it must be first transcribed into c. DNA with reverse transcriptase from Moloney murine leukemia virus or Avian myeloblastosis virus, and the c. DNA copy is then amplified.

Reverse transcriptase-PCR (RT-PCR): The technique is usually initiated by mixing short (12 -18 base) polymers of thymidine (oligo d. T) with messenger RNA such that they anneal to the RNA's polyadenylate tail. Reverse transcriptase is then added and uses the oligo d. T as a primer to synthesize so-called first-strand c. DNA. 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 Quantitative PCR: The determination or quantitation of the number of copies of a given gene achieves accurate estimation of DNA and RNA targets. n Hot-start PCR - to reduce non-specific amplification. n Multiplex-PCR n Mutagenesis by PCR. n Inverse PCR n Asymmetric PCR. n In Situ PCR. n

Polymerase Chain Reaction (PCR) Advantages of PCR: Useful non- invasive procedure. n Simplicity of the procedure. n Sensitivity of the PCR. Disadvantages of PCR: n n 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 endpoint 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: n Monitor amplification online and in real-time n Quickly and accurately quantify results n Analyze melting characteristics of PCR product n 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. Applied. Biosystem. COM Real Time PCR End point detection www. appliedbiosystems. com

The Evolution of PCR to Real-Time recent development of eal 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 n 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. n n n t(8; 21) translocation or AML 1 -ETO fusion gene t(15; 17) translocation or PML-RARA fusion gene INV(16) or MYH 11 -CBFB fusion gene t(9; 22) translocation or BCR-ABL fusion gene (p 210 and p 185 FLT 3 Mutations BCR-ABL Mutations

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. n Monitoring the MRD in leukemia and lymphoma patients by assessing PRAME (Preferentially expressed antigen of melanoma) in peripheral blood samples.

Polymerase Chain Reaction: Uses n 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 n 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. n PCR primers have also been reported for intracellular parasites like T. gondij , P. falciparum and for different strains of Trypanosoma, …. n 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 m. RNA in gastrointestinal carcinoma cell lines. * DNA analysis for genetic disease diagnosis.

Application of real time PCR in molecular diagnosis Clinical Microbiology n Viral load (HIV, HCV, HBV, …) n Bacterial load (Salmonella, Mycobacterium, . . ) n Fungal load( Candida, Cryptococcus, Aspergillus, …. ) Food microbiology n Bacterial load (Listeria, Salmonella, Campylobacter, …) Clinical Oncology n Minimal residual disease n Chromosomal translocations n Single nucleotide polymorphism (SNPs) Gene therapy n Gene transfer estimation n Biodistribution of vector Gene expression n 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 n n n n Velasco J. A new view of malignancy New York Times. April 9, 2002. . Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953; 171: 737 – 738. Pub. Med 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. Applied. Biosystem. COM Real Time PCR Watson JD, Crick FHC. Molecular structure of nucleic acids. Nature. 1953; 171: 737 – 738. [Pub. Med] 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