Question

DISCUSS THE FOLLOWING

What are the 5 key basic reagents used in PCR? Enumerate and discuss the following.

1 DNA/RNA template

2. DNA polymerase

3. Primers (forward and reverse)

4. Deoxynucleotide triphosphates (dNTPs

5. PCR buffers

Answer 1

Polymerase chain reaction, or PCR, is a laboratory technique used to make multiple copies of a segment of DNA. PCR is very precise and can be used to amplify, or copy, a specific DNA target from a mixture of DNA molecules.

The 5 key basic reagents used in PCR are: DNA/RNA template, DNA polymerase, primers (forward and reverse), deoxynucleotide triphosphates (dNTPs) and PCR buffers.

1.DNA/RNA template:

Selecting the right templates for PCR is vital as different templates are used for the various methods of PCR. For instance, when performing RT-qPCR, RNA templates are required for producing complementary DNA while DNA templates are needed for conventional PCR. It is also important to have DNA or RNA templates in high quality and quantity as this can help optimizing the efficiency of PCR. For this reason, a good PCR template preparation kit is needed for the first step of a successful PCR.

2.DNA polymerase:

All PCR reactions require a DNA polymerase which can work under high temperature (around 70 °C) as the first phase of PCR involves separation of DNA strands at high temperature (~90 °C). Taq polymerase, which is a heat-stable enzyme isolated from the thermophilic bacterium Thermus aquaticus is a commonly used DNA polymerase for PCR. Some DNA polymerases are engineered to be activated at high temperature only, so as to reduce non-specific amplification at the beginning stage of PCR.

3.Primers(forward and reverse):

Primers are short sequences of single stranded DNA that mark both ends of the target sequence. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).The initiation of DNA synthesis requires primers; short strands of nucleotides (DNA or RNA) which are complementary to the template DNA and serve as a DNA synthesis starting point for the DNA/RNA polymerase. Annealing of primers to single strand DNA requires lower temperature (50-65 °C) than the denaturation step. Once the annealing step is completed, hydrogen bonds will form between the primers and the template DNA.

4.Deoxynucleotide triphosphates (dNTPs):

Deoxynucleotide triphosphates (dNTPs) are the essential components of PCR as they act as the building blocks of nucleic acids; DNA polymerase cannot synthesize DNA without a supply of dNTPs.

5.PCR buffers:

PCR buffers ensure that the PCR reaction is conducted under optimal conditions. The major components of PCR buffer include Tris-HCl, potassium chloride (KCl) and magnesium chloride(MgCl2). Tris-HCl and KCl are responsible for maintaining a stable pH during PCR. Magnesium ions act as cofactors for DNA polymerase so as to ensure proper DNA synthesis function of the polymerase during PCR. PCR buffer is usually available at 10X concentration.