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Before the introduction of the Polymerase Chain Reaction (PCR) technique the traditional techniques of Molecular Biology that were being used for the identification and characterization of DNA fragments, were quite cumbersome and required considerable skill for the successful execution of an experiment. This is particularly true in cases where a high level of specificity and sensitivity is required, e.g. the Southern Blotting technique, introduced by Edwin Southern in 1975, fails to distinguish between pseudo-genes and the actual gene in a multi-gene family and using this technique it is also difficult to detect single copy genes.
PRINCIPLES OF THE PCR TECHNIQUE
The PCR technique is a method of amplifying nucleic acids and mimics the natural process of DNA replication in that the number of DNA molecules generated by PCR doubles after each cycle. Thus it serves as a biological photocopying machine with the added advantage that in each photocopying cycle the copied material itself becomes a template that will be photocopied in the next cycle along with the original material.
This in-vitro method of amplification of target DNA basically involves repeated cycling of three different steps (Fig. 1), which are conducted under different controlled temperatures. The first step in the PCR method is known as the denaturation step, the second as the primer annealing step and the last step in each cycle is the chain extension step. In general 25-40 of these cycles are performed in each PCR amplification using an automated DNA cycler, which controls the temperature regimen and the time spent at each temperature. At the heart of the PCR technique is the widespread adoption of the enzyme Taq polymerase, which was isolated from the bacterium Thermus aquaticus. This organism was isolated from hot springs and produces the enzyme which is highly resistant to high degrees of environmental temperatures. The enzyme currently available in the market is derived from recombinant clones which have been engineered by commercial companies, (e.g. Perkin Elmer, Roche, Biotools, Sigma etc.) to give a higher degree of specificity, accuracy and better amplification of large DNA sequences. In order to decrease spurious reactions initiated at low temperatures companies have also engineered Taq polymerases (Taq Gold® by Perkin Elmer and FastStart Taq by Roche) that will be inactive at low temperatures and will only start acting upon its template DNA after activation at a high temperature in the initial cycle. This technique known as Hot Start PCR ensures the elimination of non-specific amplification thus further increasing the specificity of the enzyme.
Denaturation: This step consists of incubating the double stranded DNA referred to as the template, at a sufficiently high temperature in order to denature it i.e. to break the bonds between the two strands to generate two single stranded DNA molecule.
Primer Annealing: The primer pairs that are involved in the annealing process (recognition & binding to the target) are short pieces of synthetic DNA (oligonucleotides), which are complimentary (perfect copy of the opposite strand) to flanking regions of the DNA to be amplified. Each primer in the pair recognizes and binds to only one strand of the target DNA, one primer in the pair is complimentary to the upstream portion of the DNA and the other to the downstream portion. The sequence of the primer is determined by the sequence of the DNA sample at the boundaries of the region to be amplified. The two DNA strands that were separated from each other in the denaturation step, remain free in solution until the temperature is lowered to the “annealing temperature” to allow the primers to anneal to the flanking regions of the target DNA.
Chain Extension Step: The third and last step in each cycle consists of the thermostable DNA polymerase mediated (5’-3’) extension of the primer-template complex. The thermostable DNA polymerase is able to synthesize (at an elevated temperature, 72oC) a complimentary copy of the initial single stranded DNA by extension of the 3’ end of each annealed primer. At this higher temperature non-specific binding of the primer is inhibited thus eliminating spurious reactions, which results in increasing the efficiency and fidelity of the reaction. At the higher temperature the enzyme also has a higher degree of turnover. Through this process the extension primers now become incorporated into the newly synthesized double stranded DNA molecule.
Repeated cycling between the above mentioned steps results in the doubling of the product after each cycle and thus an exponential increase of the product of interest, the boundaries of which are defined by the 5’ ends of the two primers. This exponential increase results because under appropriate conditions the primer extension products synthesized in cycle “n” function as templates for the other primers in cycle “n+1”.
UTILITY OF PCR
The PCR technique has had significant impact in many fields including medical genetics (diagnostic and genetic counseling), evolutionary studies (e.g. to trace human ancestry and migration patterns), developmental and forensic sciences. With the target DNA coming from a variety of sources including blood, hair, tissue, sperm, vaginal swabs of rape victims, mouth wash, biopsy and surgical specimens. The PCR technique is so powerful that only a small amount of DNA is required for the correct amplification of the target sequence, in-fact even a single molecule can rapidly produce a large amount of the specific target DNA sequence of defined length.
PCR provides a key component of molecular diagnosis. It provides a strategy for the rapid amplification of DNA (or mRNA) to search for mutations by a wide array of techniques, including DNA sequencing. Assays have been developed that allow rapid detection and characterization of a wide range of disease markers, e.g. inherited diseases like Adenosine deaminase deficiency, 1-Antitrypsin deficiency, Cystic fibrosis, Fabry disease, Familial hypercholesterolemia, G-6-PD deficiency, Hemophilia A, Hemophilia B, Lesch-Nyhan syndrome, Maple syrup urine disease, Ornithine transcarbamylase deficiency, Phenylketonuria, Sandhoff disease, sickle-cell anemia, Tay-Sachs disease, tumor cell markers, b–thalassemia, d–thalassemia and von Willebrand disease. Prenatal diagnosis for these diseases can also be done by sampling amniotic fluid or CVS.
Bacterial (MTB, Salmonella Typhi, etc.) and viral (CMV, EBV, HepA, HepB, HepC, HSV, HIV, VZV, enteroviruses etc.) genomes can also be probed in order to ascertain infection. In the case of these diseases the major advantage of PCR is that the traditional methods like ELISA probe the antibody response of the patient to the organism, whereas PCR probes the genome of the organism. Thus PCR can provide information about the infection even before the antibody response is elicited or even in its absence. Viral and bacterial genotyping can be done in order to determine therapeutic strategies. Viral load can also be calculated to determine the efficacy of therapy. Over the last few years PCR based diagnostics has been introduced in Pakistan and now is extensively relied upon by the medical community to diagnose different diseases e.g. HCV, HepB, HIV etc., and also to determine the HLA type for transplantation purposes.
Besides the diagnostic applications, which are on the increase as new disease markers are identified almost every day, PCR has significant advantage in the Medico-Legal field, where the identity of the criminal in the case of rape and murder and the identity of parent in the case of parentage cases, can be established beyond any shadow of a doubt by the use of PCR DNA fingerprinting. Fortunately in Pakistan rape cases are not that frequent and the number of illegitimate children are also quite low, as the moral fiber of the society has not yet decayed to the level of the Western societies.
Every day new disease markers are being identified that are amiable to PCR detection. There is a big debate in the scientific community that once disease and characteristic markers are identified that will enable one to identify e.g. a persons susceptibility to cancer, this data could be made use of by potential employers and insurance agents in denying job and insurance to these individuals. So who should be allowed access to this data and who should be denied it?
In addition to conducting a number of different PCR diagnostic tests, the Shifa College of Medicine PCR Lab has established for the first time in Pakistan a PCR based diagnostic facility for the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV). Before this the only SARS-CoV testing facility being offered by the National Institute of Health, Islamabad, was that of Antibody Testing for which samples are taken at day zero and then again on day 21 and sent to Centers for Disease Control and Prevention (CDC) Atlanta, USA. These tests detect antibodies produced in response to the SARS-CoV infection. Different types of antibodies (IgM and IgG) appear and change in level during the course of infection. They can be undetectable at the early stage of infection; in addition IgG usually remains detectable after resolution of the illness, thus a negative or positive Antibody titer may not necessarily reflect the actual status of infection. As apposed to this the PCR test directly detects the genetic material (RNA) of the SARS-CoV in clinical samples and thus is of more prognostic value to determine the current status of the disease. In addition a PCR can be performed in a couple of day’s time and therefore with this locally developed technologically advanced technique the patients and doctors do not have to wait indefinitely for a differential diagnosis.
PCR is a very powerful technique which has revolutionized not only Molecular Biology but also Molecular Diagnostics in the field of Medicine. The power and potential of the technique will only now be fulfilled since the complete Human Genome has been sequenced and the field of Molecular Diagnostics will completely change the way Medicine is practiced all over the World.