Polymerase Chain Reaction (PCR) is a molecular cloning method used to analyze a short sequence of DNA which is present in minimal quantities in the samples containing some genetic material. The technique has been often called ‘DNA photocopier’. Prior the invention of PCR technology, the DNA amplification was done as the segments of interest were cloned into bacterial expression vectors. As a result the DNA amplification took weeks. Now, using PCR done in test tubes, instead of cloning vectors, the whole process is completed under a couple of hours. In order to make high copy numbers of a specific DNA or RNA fragment, PCR employs a couple of standard reagents used in molecular biology labs. PCR is highly efficient to produce numerous copies of a DNA fragment.
What Is PCR Reaction?
The concept behind the PCR is quite simple, however it is a complicated process because of the many reactants involved. Usually, the DNA template concentration is initially very low, but as the reaction advances and the newly synthesized molecules become templates, the DNA fragment concentration raises significantly. Some of the reactants, such as DNA-polymerase enzyme, can become limiting. Some other reaction components, such as dNTPs and primers, are at high quantities that actually do not change during the reaction. Altering temperature and pH could result to significant changes in the dynamics of the reaction and could be to drive the process in the desired direction. To summarize, PCR is molecular cloning method with great power and versatility, which is used widely for DNA manipulation and analysis.
Some History Behind PCR Method
PCR is considered (as one of) the most significant scientific breakthroughs in the field of molecular biology. The method changed the completely the studies of nucleic acids, which was highly appreciated by the scientific community and as a result the inventor of the method – Kary B. Mullis, was honored with the Nobel Prize for Chemistry in 1993. Indeed, the PCR method drastically accelerates the pace of advancing the research of genes and genomes. All molecular and genetic analyses require significant amounts of DNA samples. Thus such studies are nearly impossible without applying PCR amplification. Now we can isolate more or less any gene from any organism using PCR.
PCR method has end up to be a foundation of genome sequencing projects. It used both for identifying DNA sequence and also for the succeeding analysis of the coding genes and their corresponding products by using high-throughput screening techniques. Once a target gene is isolated, we can use PCR to modify its sequence to enable cloning or even mutagenesis. Nowadays, PCR is a routine lab technique, which due to it’s simplicity makes possible many labs to access the power of molecular biology. Constantly new papers are published, describing new applications or new techniques of based on Polymerase Chain Reaction. In addition, numerous commercial products (including instrumentation and kits) have been launched by the industry, They can be used in various research areas, but also for PCR-based diagnostics in medicine and clinics.
How Does PCR Technology Works?
The amplification of a segment of DNA using PCR is a multi-step process. First, the DNA sample is first heated in order to denature the DNA and as a result to separate the double-stranded DNA into two single-stranded sequences. Next, a special enzyme, called Taq polymerase, using the original strands as template, synthesizes two new strands of DNA. As a result, the original DNA is duplicated, as each of the resulting two molecules contain one old and one completely newly synthesized DNA strand. Then the process can repeats in cycles – each of the strands can be used as templates to create two new copies, and so on. These cycles of denaturing DNA and subsequent synthesis of new DNA sequences, can be repeated between 30 or 40 times. At the end these iterations, lead to the multiplication of the original DNA segment into more than one billion exact copies. The entire PCR process is automated by a by a machine called a thermocycler and can be completed in a couple of hours. The PCR machine is programmed to alter the temperature of the reaction mix every few minutes and thus to regulate the cycles of DNA denaturing and DNA synthesis.
In order to fully understand what is PCR one have to read more about the RNA polymerase, the actual mechanism of the PCR reaction, the detailed PCR steps and finally the different PCR protocols and the various applications of the method.