PCR kits (also known as quantitative PCR or qPCR) is the simultaneous amplification, detection and quantification of nucleic acids using the polymerase chain reaction. qPCR is a versatile technique and a powerful tool to support your research applications.
polymerase chain reaction (PCR), is a fundamental technique that has revolutionised molecular biology. In PCR, a DNA molecule is selected and copied several times, allowing exponential amplification of the target DNA sequence.
Like DNA replication in an organism, PCR Kits requires a DNA polymerase enzyme that produces new strands of DNA by using existing strands as a template. The DNA polymerase normally used in PCR is called Taq polymerase, after the heat-tolerant bacterium from which it was isolated (Thermus aquaticus).
T. aquaticus lives in hot springs and hydrothermal vents. Its DNA polymerase is very thermostable and is most active near 70°C70°C70, °, start text, C, end text (temperature at which human or E. coli DNA polymerase would not work). Taq polymerase is ideal for PCR because of this thermal stability. As we shall see, PCR uses high temperatures repeatedly to denature the DNA template or separate its strands.
The key ingredients for a PCR reaction are Taq polymerase, primers, template DNA and nucleotides (the building blocks of DNA). The ingredients are placed in a tube, along with any cofactors needed by the enzyme, and undergo repeated heating and cooling cycles that allow DNA synthesis.
The basic steps are:
This cycle is repeated 252525 – 353535 times in a typical PCR reaction, which generally takes 222 – 444 hours, depending on the length of the DNA region being copied. If the reaction is efficient (works well), it can produce billions of copies from one or a few copies of the target region.
That’s because it’s not just the original DNA that is used as the template for each cycle. In fact, the new DNA produced in one round can serve as a template in the next round of DNA synthesis. There are many copies of the primers and many molecules of Taq polymerase floating around in the reaction, so the number of DNA molecules can almost double in each cycle. The image below shows this exponential growth pattern.
Like other DNA polymerases, Taq polymerase can only make DNA if there is a primer, a short sequence of nucleotides that provides a starting point for DNA synthesis. In a PCR reaction, the region of DNA to be copied, or amplified, is determined by the primers of the researcher’s choice.
PCR primers are short pieces of single-stranded DNA, usually about 2020-20 nucleotides long. Two primers are used in each PCR reaction that are designed to flank the target region (the region to be copied). That is, sequences are added to them that will cause them to bind to opposite strands of the DNA template only at the ends of the region to be copied. The primers bind to the template by base complementarity.
