Introduction It is not often that a technique has as immediate and dramatic an impact on science as the polymerase chain reaction (PCR) has had on molecular biology. The ability to amplify large quantities of a defined region of deoxyribonucleic acid (DNA) rapidly and specifically from a few starting copies has led to a reassessment of many old procedures, and to a host of new applications (EHRLICH, 1989; INNIS et al., 1990). Many of these are methodological, making it simpler to clone and manipulate genes. However, PCR also promises to revolutionize the diagnosis of disease. In the case of genetic disease, its claims are overwhehning, since alternatives are generally either lacking altogether, or are inadequate. However, regarding its use with mfectious diseases, particularly in developing countries, sceptics are rather more numerous. In this article I will discuss the potential strengths and weaknesses of PCR in this field. PCR is based on (i) the ability of short oligonucleotides (e.g. 15-25 bases) to hybridize rapidly and specifically to complementary DNA sequences, even in the presence of large amounts of non-complementary DNA, and (ii) the requirement that DNA polymerases have for a primer bound to a template strand in order to synthesize DNA. This means that if a suitable oligonucleotide primer is used, DNA synthesis can be specifically initiated at any point in a genome in vitro. By using 2 primers, which bind different strands of the target DNA a few hundred nucleotides apart, and allowing DNA synthesis to take place, 2 new strands of DNA will be synthesized, each of which will, crucially, include the sequences complementary to the other oligonucleotide primer. Following denaturation and reannealing steps, all new strands will themselves act as substrates for a further round of DNA synthesis, and the repetition of this cycle many times leads to an exponential increase in copies of the specific DNA target. The 3 elements of the cycle-denaturation, annealing and DNA synthesis-can all be controlled simply by temperature shifts. After the first 2 cycles, virtually all the DNA synthesized will be of a specific length, flanked by the 2 priming sequences, and the most common form of detection used is the visualization by fluorescence under ultraviolet light of a small DNA fragment of predicted size after agarose gel electrophoresis in the presence of ethidium bromide. The technical advances which have made PCR a routine tool are (i) the use of a thermostable DNA polymerase which can withstand the high temperatures required for DNA denaturation (SAIKI et al., 1988), (ii) the production of microprocessor-controlled programmable heating blocks, and (iii) the ready availability of synthetic oligonucleotides. For some years, DNA probes have promised much as diagnostic tools, but have delivered little mainly because of a lack of sensitivity. PCR has removed this barrier, although problems still remain.
Read full abstract
Jun 30, 2021
Kadiri Haritha + 1
Open Access
