: Roche Polymease Chain Reaction Salwa Hassan Teama Polymerase Chain Reaction Salwa Hassan Teama Polymerase Chain Reaction Salwa Hassan Teama


Polymerase Chain Reaction :Polymerase Chain Reaction Coping Machine for DNA Molecule


Polymerase Chain Reaction :Polymerase Chain Reaction Invented by Kary Mullis and his colleagues in the 1983. Nobel prize 1993.


Slide 4:Technique for in vitro (test tube) amplification of specific DNA sequences via the temperature mediated DNA polymerase enzyme by simultaneous primer extension of complementary strands of DNA. This system for DNA replication that allows a "target" DNA sequence to be selectively amplified, several million-fold in just a few hours.


PCR Reaction Components :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.


Template 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. The amount of template in a reaction strongly influences performance in PCR. The recommended amount of template for standard PCR is: Human genomic DNA should be up to 500 ng 1-10 ng bacterial DNA 0.1-1 ng plasmid DNA


Primers: :Primers: Typical primers are 18-28 nucleotides in length having 50-60% GC composition. Have a balanced distribution of G/C and A/T rich domains. 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. Contain no internal secondary structure. Are not complementary to each other at the 3` ends to avoid primer- dimer forming.


Buffer system: :Buffer system: 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. 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.


Deoxynucleosides triphosphate (dNTPs): :Deoxynucleosides triphosphate (dNTPs): 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.


DNA Polymerase : :DNA 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) consist of a single polypeptide chain has a molecular weight of 95 Kd, and has an optimum polymerization temperature of 70 – 80 C ?. 0.5 – 2 units/50 ? l reaction. Too little will limit the amount of product, while too much can produce unwanted non specific products.


Calculation of Melting Temperature :Calculation of Melting Temperature Tm= 2 X (number of A and T bases)+4 X (number of G and C bases). Optimal annealing temperature are 5-10 C ? lower than Tm values of the primers.


Reaction Additives :Reaction Additives In some cases, the following compounds can enhance the specificity and or efficiency of a PCR: Betadine Bovine serum albumin Dimethylysulfoxide Glycerol Pyrophosphatase Spermidine, Detergent, Gelatin,…….


Concept of PCR and Standard protocol :Concept of PCR and Standard protocol


Slide 14:All one has to do is to mix the contents in the test tube and seal the tube and then place it in a thermal cycler. The thermal cycler is programmed to cycle over and over again among three different temperatures.


Thermal Cycler :Thermal Cycler


Thermal Cycling Profile for Standard PCR :Thermal Cycling Profile for Standard PCR Initial Denaturation Each cycle includes three successive steps: Denaturation Annealing Extention


Initial Denaturation :Initial Denaturation This step consists of heating the reaction to a temperature of 94-95C ? 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. 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 results.


Slide 19: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: 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 ? .


Denaturation :Denaturation DNA is denatured into single strands


Annealing :Annealing 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. www.Roche Molecular Biochemicals: PCR Techniques


Extension :Extension Taq DNA Polymerase catalyses primer extension. 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. Roche Molecular Biochemicals


Slide 26:www.ucl.ac.uk/~ucbhjow/bmsi/lec5_images/pcr.gif


: 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 ? . The number of cycles required for optimum amplification varies depending on the amount of the starting material. Most PCR should , therefore, include only 25 – 35 cycles. As cycle increases, nonspecific products can accumulate. Following amplification, the PCR products are usually loaded into wells of an agarose gel and electrophoresed.


Gel Electrophoresis :Gel Electrophoresis 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.


Agarose Gel Electrophoresis :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.


Slide 31:Agarose Gel Electrophoresis


: 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. PCR Products


Slide 33:DNA ladder PCR product compared with DNA ladder ... PCR product compared with DNA ladder ... DNA Ladder


PCR Phases :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.


Slide 35:PCR Phases


: 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 (e.g. rare of circulating fetal cells).


Variants of PCR :Variants 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. ….


Reverse Transcriptase- PCR :Reverse Transcriptase- 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 :Reverse Transcriptase- PCR


Nested-PCR :Nested-PCR 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.


Nested-PCR :Nested-PCR


Hot Start PCR :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.


Real Time PCR :Real Time PCR 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


Evolution of PCR to Real Time :Evolution of PCR to Real Time


Slide 46: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: Uses :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………………………………………


Polymerase Chain Reaction clearly has the potential to become the routine laboratory method for diagnosis of a variety of human disorders : :Polymerase Chain Reaction clearly has the potential to become the routine laboratory method for diagnosis of a variety of human disorders : Infectious Diseases: 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: :Diagnosis of Genetic Diseases: 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.


Inherited disorders diagnosed using PCR protocols: :Inherited disorders diagnosed using PCR protocols: Alpha1 antitrypsin B thalassaemia Cystic fibrosis Duchenne muscular dystrophy Myotonic dystrophy Haemophilia A and B Huntington`s chorea Phenylketonuria Sickle cell anaemia Familial adenomatous polyposis


Prenatal Sexing and prenatal diagnosis of diseases: :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.


Slide 52:Major role in the human genome project, replacing a single polymerase with a blend of a thermostable polymerase and a proofreading (Pwo DNA polymerase) made PCR an indispensable tool in the analysis and mapping of entire genomes by greatly extending the length of the sequence that could be amplified, increasing the amount of PCR product and providing higher fidelity during PCR. 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. Combining in situ hybridization with PCR made possible the localization of single nucleic acid sequences on one chromosome within an eukaryotic organism. Amplification of archival and forensic material. Identify testing for transplantation, HLA Typing.


: Sequencing of PCR product Efficient, blunt-end cloning of any cDNA, PCR product or other dsDNA fragment PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology.


Real Time PCR : Uses :Real Time PCR : Uses 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,……..


References & Online Further Reading :References & Online Further Reading www.Roche Molecular Biochemicals: PCR Applications Manual www.Roche Molecular Biochemicals: PCR Techniques Robert F. Weaver. Molecular Biology. Fourth Edition. Page 600. McGraw-Hill International Edition. ISBN 978-0-07-110216-2 Robert F. Mueller,Ian D. Young. Emery's Elements of Medical Genetics: ISBN. 044307125X 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 Stites DP . Medical immunology. Section II. Page 270 http://en.wikipedia.org/wiki/RT-PCR http://www.ma.uni-heidelberg.de/inst/ikc/molekularbiologie/rt-pcr.jpg http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/mbglossary/mbgloss.htm http://www.dnalc.org/ddnalc/resources/electrophoresis.html


Slide 56:THANK YOU