Method & application of PCR in Molecular Biology

Abstract

The polymerase chain reaction (PCR) is a technique in moleculars biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. There are three major steps involved in the PCR technique: denaturation, annealing and extension. PCR is useful in the investigation and diagnosis of a growing number of diseases. PCR is also used in forensics laboratories. PCR can identify genes that have been implicated in the development of cancer. The present paper is an attempt to review basics of PCR in relation to its methods, application and use.

Introduction

Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions!) of a particular region of DNA. This DNA region can be anything the experimenter is interested in. For example, it might be a gene whose function a researcher wants to understand, or a genetic marker used by forensic scientists to match crime scene DNA with suspects. Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments. PCR is used in many areas of biology and medicine, including molecular biology research, medical diagnostics, and even some branches of ecology. . Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory research for a broad variety of applications including biomedical research and criminal forensics. It is a common tool used in Pharmaceutical Biotechonology. PCR is a quick and easy method for generating unlimited copies of any fragment of DNA. From the daily practicalities of medical diagnosis to the theoretical framework of systematics, from courts of law to field studies of animal behavior, PCR takes analysis of tiny amounts of genetic material - even damaged genetic material to a new level of precision and reliability. Furthermore, many important contributions to the development and application of PCR technology have been made.

Principle of PCR

The basic PCR principle is simple. It is a chain reaction and one DNA molecule is used to produce two copies, then four, then eight and so forth. Thiscontinuous doubling is accomplished by specificproteins known as polymerases, enzymes that areable to string together individual DNA buildingblocks to form long molecular strands. To dotheir job polymerases require a supply of DNAbuilding blocks, i.e. the nucleotides consisting ofthe four bases adenine (A), thymine (T), cytosine(C) and guanine (G). They also need a smallfragment of DNA, known as the primer, to whichthey attach the building blocks as well as a longerDNA molecule to serve as a template forconstructing the new strand. If these threeingredients are supplied, the enzymes willconstruct exact copies of the templates. PCR is amethod used to acquire many copies of anyparticular strand of nucleic acids. Methods: In molecular biology, real-time polymerase chain reaction, also called quantitative real time polymerase chain reaction is a laboratory technique based on the PCR, which is used to amplify and simultaneously quantify a targeted DNA molecule. Traditionally, PCR is performed in a tube and when the reaction is complete the products of the reaction (the amplified DNA fragments) are analyzed and visualized by gel electrophoresis. However, Real-Time PCR permits the analysis of the products while the reaction is actually in progress. This is achieved by using various fluorescent dyes which react with the amplified product and can be measured by an instrument. This also facilitates the quantitation of the DNA. Quantitative PCR (Q-PCR), as this technique is known, is used to measure the quantity of a PCR product (usually in a real-time PCR procedure).

PCR Applications

PCR is helping in the investigation and diagnosis of a growing number of diseases. It has also long been a standard method in all laboratories that carry out research on or with nucleic acids. Even competing techniques such as DNA chips often require amplification of DNA by means of PCR as an essential preliminary step. The polymerase chain reaction is used by a wide spectrum of scientists in an ever-increasing range of scientific disciplines. The use of reverse transcriptase’s to evaluate RNA levels and the extension of PCR technology to quantify DNA amplification in real time has brought major advances to the application of PCR. By allowing the determination and quantification of changes in gene expression, these techniques have provided a greater understanding of disease processes and now serve as a foundation for diagnostics and basic science research. In microbiology and molecular biology, for example, PCR is used in research laboratories in DNA cloning procedures, Southern blotting, DNA sequencing, recombinant DNA technology, to name but a few. PCR can also be employed with significant precision to predict cure of the disease. Molecular cloning has benefited from the emergence of PCR as a technique.

Conclusion

The advancement of science has transformed our lives in ways that would have been unpredictable just a half-century ago. Molecular methods have shown a promise in this aspect. PCR and its applications hold scientific and medical promise. PCR has very quickly become an essential tool for improving human health and human life. PCR has completely revolutionized the detection of RNA and DNA viruses and is also valuable as a confirmatory test. PCR is a rapid technique with high sensitivity and specificity. PCR has also been credited to have been able to detect mixed infections with ease in many studies. PCR, a more sophisticated technique, requires infrastructural support, is expensive but nevertheless, one cannot discount its utilitarian advantages which are many compared to the existing conventional diagnostic methods.