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Polymerase Chain Reaction (PCR): a Short Review

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Polymerase Chain Reaction (PCR): a Short Review REVIEW ARTICLE Polymerase Chain Reaction (PCR): A Short Review *MT Rahman1, MS Uddin2, R Sultana2, A Moue3, M Setu4 *Prof. Dr. Md. Tahminur Rahman, Professor & Head, Pathology, Anwer Khan Modern Medical College, Dhaka 2Muhammed Salah Uddin, Senior Research Assistant, ICDDRB, Mohakhali, Dhaka 2Razia Sultana, Senior Research Assistant, ICDDRB, Mohakhali, Dhaka 3Arumina Moue, Mhil Student(Microbiology), Stamfored University Dhaka 4Dr. Muntahina Setu, Asstt Profe, Dept. of Paediatric, Anwer Khan Modern Medical College, Dhaka *Corresponding author ABSTRACT Diagnosis of disease now a days is mostly laboratory dependent. Due to recent advances in medical science and molecular biology, most of the diagnosis of uncommon, complicated, unusual presentation of disease has left the option of molecular diagnosis as the number one diagnostic modalities. Many molecular techniques are now being widely used throughout the world including PCR, flow cytometry, tissue microarray, different blots, and genetic diagnosis. Among these PCR is the most widely accepted, commonly used diagnostic modalities with very high specificity and sensitivity for correct diagnosis. We have reviewed the principle, application, advantages and disadvantages of PCR in laboratory diagnosis of disease. Key words: PCR, Molecular techniques, Review Introduction Polymerase chain reaction (PCR) is a new, was awarded the Nobel prize in Chemistry along 2 popular molecular biology technique for with Michael Smith for his work on PCR . The enzymatically replicating DNA without using a PCR is commonly carried out in a reaction living organism, such as E. coli or yeast. The volume of 10-200 ml in small reaction tubes technique allows a small amount of the DNA (0.2-0.5 ml volumes) in a thermal cycler. The molecule to be amplified many times, in an thermal cycler heats and cools the reaction tubes exponential manner. With more DNA available, to achieve the temperatures required at each step analysis is made much easier. PCR is commonly of the reaction. Many modern thermal cyclers used in medical and biological research labs for make use of the Peltier effect, which permits a variety of tasks, such as the detection of both heating and cooling of the block holding hereditary diseases, the identification of genetic the PCR tubes simply by reversing the electric fingerprints, the diagnosis of infectious diseases, current. Thin-walled reaction tubes permit the cloning of genes, paternity testing, and DNA favorable thermal conductivity to allow for rapid computing1. The technique was developed in thermal equilibration. Most thermal cyclers have 1983 by Kary Mullis, PCR is now a common heated lids to prevent condensation at the top of and important technique used in medical and the reaction tube. Older thermocyclers lacking a biological research labs for a variety of heated lid require a layer of oil on top of the applications. These include DNA cloning for reaction mixture or a ball of wax inside the tube. sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of Uses of PCR hereditary diseases; the identification of genetic PCR can be used for Diagnosis of many human fingerprints (used in forensic sciences and diseases, broad variety of experiments and paternity testing); and the detection and analyses3,4,5. Some examples are discussed diagnosis of infectious diseases. In 1993, Mullis below. AKMMC J 2013: 4(1): 30-36 Polemerase Chain Reaction (PCR): A Short Review 31 1. Infectious diseases HIV, CMV, Mycoplasma, The PCR reaction is carried out in a thermal Pneumonia, Cancer, Syphilis, fungal & cycler. This is a machnie that heats and cools Protozoal disease, hepatitis etc. the reaction tubes within it to the precise 2. Diagnosis of cancer specially leukaemia and temperature required for each step of the lymphomas reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100 l per 3. Genetic fingerprinting, paternity test tube), a heated lid is placed on top of the PCR permits early diagnosis of malignant reaction tubes or layer of oil is put on the diseases such as leukemia and lymphomas, surface of the reaction mixture. which is currently the highest-developed in cancer research and is already being used Primers routinely. PCR assays can be performed directly The DNA fragment to be amplified determined on genomic DNA samples to detect by selecting primers. Primers are short, translocation-specific malignant cells at a artificial DNA strands not more than fifty sensitivity that is at least 10,000-fold higher than (usually 18-25 bp) nucleotides that are that of other methods. complementary to the beginning and end of the PCR also permits identification of non- DNA fragment to be amplified. They anneal cultivatable or slow-growing microorganisms (adhere) to the DNA template at the starting and such as mycobacteria, anaerobic bacteria, or ending points, where the DNA-Polymerase viruses from tissue culture assays and animal binds and begins the synthesis of the new DNA models. The basis for PCR diagnostic strand applications in microbiology is the detection of The PCR process consists of a series of twenty infectious agents and the discrimination of non- to thirty-five cycles. Each consists of three pathogenic from pathogenic strains by virtue of steps. specific genes. 1. The double-stranded DNA has to be heated to PCR is used to amplify a short, well-defined 94-960C in order to separate the strands. This part of a DNA strand. This can be a single step is called denaturing; it breaks apart the gene, or just a part of a gene. As opposed to hydrogen bonds that connect the two DNA living organisms, the PCR process can copy strands. Prior to the first cycle, the DNA is only short DNA fragments; usually up to 10 kb often denatured for an extended time to ensure (kb stands for kilo base pairs). Certain methods that both the template DNA and the primers can copy fragments up to 40 kb in size, which is have completely separated and are now single- still much less than the chromosomal DNA of a strand only. Time 1-2 minutes up to 5 minutes. eukaryotic cell -for example, a human cell Also Taq-polymerase is activated by this step. contains about three billion base pairs. 2. After separating the DNA strands, the PCR requires several basic components. temperature is lowered so the primers can attach These components are: themselves to the single DNA strands. This step n DNA template, or cDNA which contains the is called annealing. The temperature of this region of the DNA fragment to be amplifie stage depends on the primers and is usually 50C below their melting temperature (45-600C). A n Two primers, which determine the beginning wrong temperature during the annealing step can and end of the region to be amplified (see result in primers not binding to the template following section on primers) DNA at all, or binding at random. Time 1-2 n Taq polymerase, which copies the region to minutes. be amplified 3. Finally, the DNA-Polymerase has to fill in n Nucleotides, from which the DNA-Polymerase the missing strands. It starts at the annealed for new DNA primer and works its way along the DNA n Buffer, which provides a suitable chemical strand. This step is called extension. The environment for the DNA-Polymerase. extension temperature depends on the DNA- 32 AKMMC J 2013: 4(1) MT Rahman, MS Uddin, R Sultana, MA Mumu et al Polymerase. The time for this step depends both Practical modifications to the PCR technique: on the DNA-Polymerase itself and on the length Nested PCR- Nested PCR is intended to reduce of the DNA fragment to be amplified. As a rule- the contaminations in products due to the of-thumb, 1 minute per 1 kbp. amplification of unexpected primer binding Although these resulting 'fingerprints' for sites. Two sets of primers are used in two example, parent-child or sibling, can be successive PCR runs, the second set intended to determined from two or more genetic amplify a secondary target within the first run fingerprints, which can be used for paternity product. This is very successful, but requires tests. A variation of this technique can also be more detailed knowledge of the sequences used to determine evolutionary relationships involved. between organisms. The PCR product can be Inverse PCR- Inverse PCR is a method used to identified by its size using agarose gel allow PCR when only one internal sequence is electrophoresis. Agarose gel electrophoresis is a known. This is especially useful in identifying procedure that consists of injecting DNA into flanking sequences to various genomic inserts. agarose gel and then applying an electric current This involves a series of digestion and self to the gel. As a result, the smaller DNA strands ligation before cutting by an endonuclease , move faster than the larger strands through the resulting in known sequences at either end of the gel toward the positive current. The size of the unknown sequence. PCR product can be determined by comparing it with a DNA ladder, which contains DNA RT-PCR (Reverse Transcription PCR) is the fragments of knows size, also within the gel. method used to amplify, isolate or identify a known sequence form a cell or tissues RNA The detection of hereditary diseases in a given library. Essentially normal PCR preceeded by genome is a long and difficult process, which transcription by Reverse transcriptase (to can be shortened significantly by using PCR. convert the RNA to cDNA) this is widely used Each gene in question can easily be amplified in expression mapping, determining when and through PCR by using the appropriate primers where certain genes are expressed. and then sequenced to detect mutations. Viral diseases, too, can be detected using PCR Asymetric PCR-Asymetric is used to through amplification of the viral DNA.
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