PCR Master Mix

The polymerase chain reaction (PCR) is used to amplify a specific fragment of DNA strand from a complex mixture of initial starting material. For setting up a PCR, you need to prepare a master mix. Generally, the PCR master mix consists of a target double-stranded DNA template, two oligonucleotide primers which hybridize to bordering sequences on either strands of the template, all four deoxyribonucleoside triphosphates (dNTPs) and a DNA polymerase.

Generally, you need to prepare 10-200µl reaction volume in small reaction tubes (200-500µl volume tubes) for setting up a PCR. The PCR is carried out in a thermal-cycler. Thermal-cycler is a machine that provides varying temperatures by heating or cooling the reaction tubes, as per the requirement of each reaction step. It is preferable if you use thin-walled reaction tubes, since it facilitates rapid thermal equilibration by maintaining proper thermal conductivity.

Now-a-days, in most of the thermal-cyclers, heated lids are there. As a result of this, the evaporating reaction mixture does not condense at the top of the reaction tube and remain within the reaction volume, maintaining the original composition. In thermal-cyclers without heated lids, a layer of mineral oil is added on the top of the reaction mixture to prevent evaporation. Alternatively, a wax ball can be inserted inside the reaction tube.

Once you have selected the appropriate target substrate, you need the following basic components for setting up the reaction

1)    Target DNA

You can obtain DNA for initial amplification from different types of sources. The template DNA source can be plasmid DNA, genomic DNA, cDNA, prokaryotic cells or even eukaryotic cells. You just need to simply boil prokaryotic or eukaryotic cell samples for extracting the DNA for PCR.

The minimal amount of template DNA required for PCR depends on the source. For example, 1µg of DNA is required if isolated from mammalian cells, whereas 1ng of DNA is sufficient enough if the source is bacteria. However, in case of plasmid DNA, you just need as little as 1pg of DNA for PCR amplification.

Double-stranded DNA (e.g. plasmid, genomic DNA etc.) as well as single-stranded DNA (e.g. cDNA) can be used as template DNA. Amplifications can be obtained both from circular-coiled DNA and linear DNA. However, it has been found that amplification from circular-coiled DNA is less efficient as compared to linear DNA. The main reason behind this is the huge and complex structure of circular-coiled DNA, which hinders in the proper binding of the primers to the template DNA, resulting in poor amplification. With most of the common PCR methods, DNA fragments of up to ~10kb can be easily amplified. However, using specialized techniques even the fragments up to 40kb can be amplified.

2)    Primers

The most crucial factor that decides the efficiency and specificity of the PCR amplification is the primers. Primers are the short stretches of oligonucleotides that anneals to the template DNA to amplify the fragment. They are essential for PCR amplifications because DNA polymerases can only bind the new nucleotides to the existing oligonucleotide/strand in 5’ to 3’ direction. Thus, primer hybridized to the template DNA serves as a short DNA strand to which the DNA polymerases can easily keep on adding new nucleotides to copy the existing DNA strand.

The primers are designed complementary to the DNA sequences bordering the target sequence to be amplified. But at the same time you must check that the forward and reverse primers are not complementary to each other, otherwise they will bind to each other to form primer dimers. This will obstruct proper DNA amplification, since no primer population will be left to anneal to the template DNA.

The other parameters that you need to keep in your mind while designing your primers are primer length, GC content and the melting temperatures. The ideal primer length is 20-28mer. The appropriate GC content is 40-60% and you should try to keep melting temperature in the range of 55-65ºC. There are many bioinformatics tools available with the help of which you can design your primers. Some examples are Oligo calculator, Generunner, Primer3, GeneFisher etc.

While designing primers the three objectives that you need to keep in your mind are:

i) The primers must be designed in such a way that there is high yield of the desired product.

ii) Amplification of unwanted non-specific sequences should be avoided.

iii) Subsequent manipulation of the amplified product shall be achieved easily.

3) dNTPs

Equimolar concentrations of dATP, dTTP, dCTP and dGTP must be present in the standard PCR. If you are using 1.5mM MgCl₂ along with the Taq DNA polymerase in 50µl reaction, then you should add 200-250µM of each dNTP. Higher concentrations of dNTPs should be avoided, since it reduces the yield by quenching the Mg²⁺ ions which are essential for proper activity of polymerases. Also, there will be more probability of incorporation of mismatched nucleotides by the polymerase.

Now-a-days, stocks of varying concentrations (10, 25 or 100mM) of dNTPs are available commercially. You should store stocks of these dNTPs at -20ºC. These stocks are provided with pH 8.1, which minimizes its damage from freeze and thaw. However, it would be preferable if you store these stocks in small aliquots, as this will prevent dNTPs from degradation by repeated freezing and thawing.

4) DNA polymerase

DNA polymerase is an enzyme needed for the synthesis of the DNA fragment. For PCR, a thermo-stable DNA polymerase is required, so that it can withstand higher temperatures. DNA polymerase needs a short stretch of oligonucleotides for the synthesis of new DNA fragment.

The polymerase gets attached to the primer-template hybrid and synthesis of new DNA strand begins using primers as a starting point. It keeps on adding single free nucleotides one by one to the exposed 3′-hydroxyl group provided by the primer. The DNA synthesis occurs in 5′ to 3′ direction only and not in 3′ to 5′ direction. This is because the nucleotides can be added only to the 3′ end and not to the 5′ end of the nucleic acid by DNA polymerase.

Now-a-days, a wide range of enzymes are available commercially. You can choose among them on the basis of their reliability, efficiency and ability to synthesize large fragments in accordance to your needs. IfTaq polymerase is used, then generally its 2-5 units are added in 50µl reaction volume. If higher amount of enzyme than this level is taken, then it may lead to accumulation of non-specifically amplified PCR product and there will be low yield of desired fragment. If accurate and flawless amplification of the DNA fragment is desired, then proofreading DNA polymerases are also available. For Example, Pfu DNA polymerase, Vent DNA polymerase, Phusion DNA Polymerase, etc. They remove mismatched nucleotides, if any, during amplification by their 3′ to 5′ exonuclease activity.

5) Standard Reaction Buffer

Buffer is the most basic component of the reaction master mix. It provides the platform for the reaction to take place. The main function of the buffer is to maintain the pH so as to make the reaction feasible. Any change in the PCR buffer will affect the consequence of the amplification. Basically, standard buffer consists of Tris-Cl at a concentration of 10mM.

During PCR cycling, when the temperature reaches 72ºC, there is a dip of more than a full unit in the pH of the reaction mixture. The optimal pH for the reaction mixture is ~7.2. Therefore, the pH of the standard reaction buffer is kept approximately 8.3-8.8, so that during the cycling process the pH of the reaction mixture may become desirable after getting reduced.

Buffer also contains KCl and MgCl₂ as the source of monovalent and divalent cations.

i) Mono-valent Cations

You need appropriate amount of mono-valent cations in the standard reaction buffer for primer annealing and for proper amplification of the DNA fragments. Generally, KCl is added as the source of mono-valent cations. Generally, 50mM KCl is sufficient enough to amplify larger DNA fragments. However, if the target DNA fragment is shorter, then higher concentration of KCl (70-100mM) would be suitable for the reaction.

ii) Di-valent Cations

Presence of free divalent cations plays a critical role in exhibiting proper activity by all thermo-stable DNA polymerases. You will find MgCl₂ as the most common source of divalent cations in most of the commercially available standard reaction buffers. Sometimes MnCl₂ is used alternatively but polymerases work less efficiently in this case. Generally, 1.5mM MgCl₂ is used in standard reaction buffers. Its higher concentration results in the production of non-specific amplified products, whereas inadequate amount reduces the yield.

Now-a-days, stock solutions of buffers are supplied along with the DNA polymerase. These buffers are optimized for particular type of DNA polymerase. For example, Taq polymerase is supplied with 10X standard Taq buffer. Similarly, different DNA polymerases are supplied with their respective buffers.