Polymerase chain reaction or PCR is the in-vitro technique for the synthesis of the DNA, that is, it is an in-vitro DNA replication procedure. This technique is generally used in case of molecular biology for the amplification of a single copy or a few copies of DNA segments. After the completion of this procedure, it leads to the generation of thousands or millions of copies of that particular DNA segment from very few copies of DNA. This PCR technique possesses an elegant simplicity.
COMPONENTS WHICH ARE REQUIRED IN PCR
- DNA template containing the target region(s) which is/are going to be amplified.
- Two primers of DNA (forward primer and reverse primer). The synthetic oligonucleotides (primers, are prepared) should be complementary to the sequences on the opposite strands of the target DNA at positions defining the ends of the segment to be replicated. These primers basically serve as the replication primers.
- A DNA polymerase enzyme. The primers are extended by this enzyme and the enzyme polymerizes the new DNA strand. In PCR, since a high temperature is required, so, for the polymerization of new DNA strands, thermophilic DNA polymerases are used. As for example: Taq DNA polymerase, Deep vent R DNA polymerase.
- Deoxynucleoside triphosphates or dNTPs (dATP, dGTP, dCTP and dTTP). They act as bricks or as the building blocks in this procedure.
- A buffer solution to maintain the appropriate chemical environment. This is required for the optimum polymerase enzymatic activity and its stability.
- Mg2+, a bivalent cation.
Thermal cycling is involved in this polymerase chain reaction. This thermal cycling consists repeated heating and cooling cycles of the reaction for melting and enzymatic replication of the DNA. It sets a chain reaction motion in which exponential amplification of the DNA template takes place.
The isolated DNA which contains the segment to be replicated, is first heated briefly to denature it. After heating it, it is then cooled in the presence of the large excess of the synthetic oligonucleotide primers. After that, 4 dNTPs along with the Taq DNA polymerase are added to it and the primed DNA segment is replicated selectively in vitro. The cycle of the heating, cooling and replication is repeated 25 to 30 times over a few hours in an automated process. The basic three steps of the PCR are: Denaturation (94-98°C); Annealing (50-65°C) and extension (72°C). This whole process can be described through the below diagram.
Importance of PCR
This powerful technique is used in case of the (i) Medical diagnosis, (ii) Forensics, and (iii) The studies of molecular evolution. Valuable diagnostic information can be provided by the PCR in medicine. Bacteria and viruses can be readily detected with the use of specific primers. As for example: certain cancers are early detected by the promising PCR method. Mutations of certain growth-control genes (ras-genes) can be identified as well by the PCR, along with genetic testing, tissue typing, identifying oncogenes, etc. In addition to all of these, PCR is used as an indispensible tool in forensic science, particularly in DNA fingerprinting, Genetic mapping, genetic testing, tissue typing etc.
Let’s take a look at a type of question you can expect in GAMSAT from this topic.
A) The separation of the DNA strand is caused due to the:
- Presence of the DNA helicase enzyme (present within the cell itself) along with the high temperature.
- Presence of the DNA gyrase enzyme.
- Hydrogen bond destruction.
- All of the above
2. The enzyme which cuts DNA at a specific recognition nucleotide sequences or near that specific recognition nucleotide sequences (restriction sites) is known as restriction enzyme or restriction endonuclease. Restriction map is a diagram of a DNA molecule by which the relative positions of the cleavage sites of various restriction enzymes are shown. The DNA is subjected to digestion with two or more restriction enzymes both individually and mixtures to generate the restriction map. After that, the fragment lengths in the various digests are compared by their electrophoretic mobilities with respect to standards of known molecular masses and the restriction map is constructed.
The below unknown plasmid is subjected to digestion with the two restriction enzymes EcoRI and BamH I both individually and in mixtures. After that, the resulting components are run in a agarose gel. The picture of the agarose gel is also shown below.
B) Which of the following statement(s) can be concluded from the above gel picture?
- The correct sizes of the digested DNA sequences are not amplified properly
- The DNA primers are not complementary to the target DNA strand
- Both A and B
- None of the above