• IGHODARO OGHOGHO UYIOSA

Polymerase chain reaction (PCR): Evaluation of different optimization parameters for appropriate PCR process.

Abstract

PCR is a method used to produce relatively large amounts of a specific DNA sequence. The productivity of PCR method depends on different reaction conditions such as the concentration of the DNA template, concentration of magnesium ions, DNA template dilution and polymerase concentration. The aim of this study was to find optimal reaction conditions required for appropriate PCR process. To check the correct conditions the agarose gel and polyacrylamide gel electrophoresis were used. One single, strong amplified band and no unspecific product describe the most suitable amount of given conditions. In the results of this experiment, the most suitable annealing temperature was 62^oC, the most proper amount of concentration of magnesium was 2nMol, and the best template dilution was 2µl. The important parameters evaluated in this experiment were template optimisation, annealing temperatures and magnesium chloride concentrations with and without touchdown PCR.

1. Introduction

PCR is a simple, enzymatic assay, which allows for the amplification of a specific DNA fragment from a complex pool of DNA. PCR can be done using source DNA from a variety of tissues and organisms, including peripheral blood, skin, hair, saliva, and microbes. Only trace amounts of DNA are needed for PCR to generate enough copies to be analysed using conventional laboratory methods. For this reason, PCR is a sensitive assay (Lilit andNidhi 2013).

To carry out a PCR it is essential to have the following reagents: DNA template, forward and reverse primers, PCR buffer, magnesium chloride (MgCl₂), dNTP and DNA Taq polymerase.

The annealing temperature allows for the annealing of the primers to the single stranded DNA. It depends on the length and composition of the primers. If the temperature is too high, then the primers will not anneal correctly, and if the annealing temperature is too low then the primers will anneal non-specifically (Hecker et al. 1996).

Magnesium ions interact with the DNA polymerase enzyme during this process. The magnesium ion interacts with negatively charged molecules in the reaction. Positive ions of magnesium interact with the negatively charged DNA strands to mask the forces of repulsion (Markoulatos et al. 2002).

Template DNA is a fragment of DNA which is needed to create required copies.

DNA Taq polymerase is a polymerase enzyme, which is essential for DNA replication, this means that DNA polymerase synthesizes DNA molecules from their nucleotide building blocks (Huang et al. 1992). The nucleotides include the four bases – adenine, thymine, cytosine, and guanine (A, T, C, G) – that are found in DNA. These act as the building blocks that are used by the DNA polymerase to create the resultant PCR product.

During this experiment, two methods were used to visualise the PCR products formed. These were Polyacrylamide gel electrophoresis (PAGE) and Agarose gel electrophoresis method.

Electrophoresis is a separation procedure which is based on the separation of DNA fragments by size, shape and charge. The clue of this process is the mobility of ions in an electric field (nucleoid acids, which are negatively charged migrate to the anode – positive electrode) (Stellwagen, 1998).

Aim

The aim of this experiment was to assess which parameters as annealing temperature, concentration of magnesium, and template DNA influence DNA amplification efficiency and specificity.

2.0 Materials and methods

This section was divided into two parts. In part A, a PCR reaction 1 set-up using optimised PCR mastermix was done, while in part B, a PCR reaction was set-up to test four different variables to optimise a PCR reaction.

2.1 Part A

Setting up the PCR reaction

A mastermix enough for four reactions was made. 30µl of H₂O, 50µl of 2X PCR mastermix and 4µl each of forward and reverse primers were pipetted and mixed in an Eppendorf tube from which, 24µl of the mastermix was pipetted into three separate PCR tubes (i.e. tube 1, 2, and 3). 1µl of sample DNA, 1µl of 1/10 diluted DNA and 1µl of H₂O were added to each tubes respectively and each amplified on a PCR block running the following programme:

Denaturing step done at 94^oC for two minutes

Amplification step done at 94^oC for 30 seconds, 55^oC for 30 seconds and 72^oC for 1 minute. This step was repeated for 35 cycles.

Finally, the extension step was done at 72^oC for 3 minutes.

Agarose gel electrophoresis (2% agarose gel for PCR)

An agarose gel was submerged in a gel tank filled with TBE buffer. Then 5µl of gel loading buffer was added to each sample and mixed. Next, the first well was loaded with the molecular weight marker and then 10µl of each sample was loaded into each respective wells. Next, the gel was run for 45 minutes at 80V.

Finally, the gel was visualised on the gel documentation system to show separation and migration of the DNA.

2.2 Part B

PCR optimisation reaction

For this protocol, PCR reactions were set up and individual components of the reactions were varied in other to optimise them as follows:

Annealing temperature optimisation

17.75µl of water, 2.5µl of 10x reaction buffer, 0.75µl of Magnesium Chloride (50mMol), 0.5µl of dNTPs, 1µl each of forward and reverse primers, 0.5µl of Taq polymerase and 1µl of DNA were pipetted into 5 separate tubes (i.e. tube 1, 2, 3, 4, 5) which were then placed on a gradient PCR block, with one tube at each of the following temperature 46^oC, 52^oC, 55^oC, 58^oC, and 65^oC. Next, the PCR block was set to the following programme:

Denaturing step at 94^oC for two minutes

Amplification step at 94^oC for thirty seconds, 46-65^oC for thirty seconds, and 72^oC for one minute. This stage was repeated for thirty-five cycles.

Finally, the extension step was done at 72^oC for three minutes.

Template optimisation

A PCR mastermix containing 13.75µl of water, 2.5µl of 10x reaction buffer, 0.75µl of 50mMol magnesium chloride, 0.5µl dNTPs, 1µl each of forward and reverse primer and 0.5µl of Taq polymerase were pipetted into five tubes. Next, 5, 2, 1, 0.1, and 0.01µl of template DNA and, 0, 3, 4, 4.9. 4.99µl of H₂O were added to each tubes respectively. Next, the tubes were then amplified on a PCR block using the same cycle parameters as set out in part A.

Magnesium concentration

A PCR mastermix mastermix containing 16µl of water, 2.5µl of 10x reaction buffer, 0.5µl dNTPs, 1µl each of forward and reverse primer, 0.5µl of Taq polymerase and 1µl of DNA were pipetted into five tubes (i.e. tube 1, 2, 3, 4, and 5). Next, a Mgcl₂ dilution was prepared to get a final Mgcl₂ mMol of 0.5, 1.5, 2, 3 and 5mMol which were then added to each tubes respectively to give a final volume of 25µl. Next, the tubes were then amplified on a PCR block using the same cycle parameters as set out in part A.

Touchdown PCR

A PCR mastermix was prepared using the same variables outlined for magnesium concentration.

Then the touchdown PCR programme used was as follows:

94°C for 3 minutes then

94°C for 30 seconds, 64°C for 30 seconds and 72°C for one minute for three cycles,

94°C for 30 seconds, 61°C for 30 seconds, and 72°C for one minute for three cycles,

94°C for 30 seconds, 58°C for 30 seconds, and 72°C for one minute for three cycles,

94°C for 30 seconds, 55°C for 30 seconds, and 72°C for one minute for three cycles,

94°C for 30 seconds, 53°C for 30 seconds, and 72°C for one minute for thirty cycles, and finally 72°C for three minutes.

Sample preparation and polyacrylamide gel electrophoresis

After all the different optimisation protocols, the samples to be loaded into the gel were prepared by adding 5µl of loading buffer to each PCR reaction and mixed. Next, 5µl of DNA ladder was pipetted into the first well while 10µl of sample were pipetted into each designated wells. The gel was then run at 100V for 45 minutes. After which, the gel was recovered and placed in a weighing boat containing 1x TBE buffer. Next, 5µl of ethidium bromide was carefully introduced into the weighing boat and left for 10 minutes before visualisation with a gel documentation system.

3.0 Result

PCR reactions were set up in 5 different PCR tubes with all the required components for complete PCR reaction , save for a particular factor which was varied to ascertain the optimal concentration necessary for the production of the highest amount of pure specific product. The results obtained from these various optimised factors are represented in the gels below.

Part A

Optimized PCR

1µl of DNA template was loaded in the well labelled neat DNA and it revealed the highest amount of product formed, the well containing a 1/10 diluted DNA had a much lower amount of product formed while the well with the water blank yielded no product. Also, unspecific products were not formed.

Figure 1: – Optimised PCR agarose gel.

Part B

The results of different PCR optimisation reactions

PCR annealing temperature optimisation

Figure 2: – PCR annealing temperature optimisation polyacrylamide gel.

Key: L-molecular weight ladder, lane 1- 48°c, lane 2- 52°c, lane 3- 55°c, lane 4- 62°c, lane 5- 65°c, unspecific product, specific product.

From Figure 2 above, there are three unspecific products formed in lane 1, a greater specific product with insignificant unspecific product formed in lane 2, insignificant unspecific products formed in lane 3 and there is also a decrease in the intensity of the specific product formed compared with that of lane 2. While in lane 4, there is a minor decrease in the intensity of the specific product formed thus, the intense quantity of the specific product formed (i.e. there is a strong amplification here) and in lane 5, there is a major decrease in the quantity of specific product formed. Therefore, the quantity of the product decreases as the quantity of the template DNA decreases, and the quantity of the specific and unspecific products increases as the quantity of the template DNA increase.

Template dilution optimisation

Figure 3: Template dilution optimisation polyacrylamide gel.

Key: – L -molecular weight ladder, lane 1- 5µl, lane 2- 2µl, lane 3- 1µl, lane 4- 0.1µl, lane 5- 0.01µl template DNA, specific product, unspecific product.

From the gel above, there are three unspecific products formed in lane 1 and also the intensity of the specific product formed is high, the unspecific product formed in lane 2 is insignificant and the specific product formed is greater in intensity (i.e. has a higher amplification) compared with that of lane 1, in lane 3 and 4, the intensity of the specific product formed decreased compared with that of lane 2, while in lane 5, there is a major decrease in the intensity of the specific product formed. Therefore, the quantity of the product decreases as the quantity of the template DNA decreases, and the quantity of the specific and unspecific products increases as the quantity of the template DNA increase.

Magnesium dilution optimisation

Figure 4: Magnesium dilution optimisation polyacrylamide gel.

Key: – L -molecular weight ladder, lane 1- 0.5mmol, lane 2- 1.5mmol, lane 3- 2mmol, lane 4- 3mmol, lane 5- 5mmol, specific product, unspecific product.

Form the gel above, there is an unspecific and a specific product formed in lane 1, in lane 2 there is a decrease in the intensity of the unspecific product formed and also there is an increase in the amount of specific product formed. In lane 3, there were no production of unspecific products and the intensity of the specific product formed remained high. In lane 4, there is evidence of the presence of an unspecific product formed but the intensity of the specific product formed remained high. While in lane 5, there is a minor reduction in the intensity of the specific product formed while there is visible presence of formation of unspecific products.

Magnesium touchdown optimisation

Figure 5: –Touchdown magnesium concentration optimisation.

Key: – L – molecular weight ladder, lane 1- 0.5 mMol, lane 2- 1.5 mMol, lane 3- 2mMol, lane 4- 3 mMol, lane 5- 5mMol, unspecific product, specific product.

From the gel above, in lane 1, the intensity of the unspecific product formed is the same with that of the specific product formed. While in lane 2, 3 and 4, the intensity of the specific products formed are the same while the intensity of the unspecific products gradually decreased. There was no unspecific product formed in lane 5, however there was a reduction in the intensity of the specific product formed compared to that of lane 4.

Discussion

This experiment was performed to evaluate different optimisation protocols to optimise PCR reactions.

For the PCR reaction using an optimised PCR mastermix in part A, the highest amount of products formed was observed in the well containing 1µl of DNA template.The annealing temperature is the most important optimisation, because it can have an influence on the specificity of the reaction. If the temperature is too high, the hybridization will not take place thus, templates and primers remain dissociated. If the temperature is too low, mismatched hybrids will occur. Correct annealing temperature must be low enough to start hybridization between template and primer, and also high enough to prevent forming mismatched hybrids (Roux, 2009). According to the results from annealing temperature optimization polyacrylamide gel (Figure 2), the most suitable annealing temperature was 62^oC, because the band was clear and single as opposed to the 48^oC, 52^oC and 55^oC, where the smears (i.e. unspecific products) were shown. The intensity of the band in 62^oC was the strongest compared with that of 65^oC.

The most suitable template dilution for PCR was 2µl, because it gave in the polyacrylamide gel in Figure 3, the most bright, single band with very low amount of unspecific products formed.

Besides, annealing temperature and template dilution parameters, PCR reaction components could also lead to non-specific amplification. Two variables, which are reported to greatly influence the specificity of the PCR reaction, are magnesium and dNTP concentration (Dwivedi et al. 2003).

For magnesium dilution touchdown (Figure 5), the molarities at 1.5, 2 and 3mMol showed very similar amount of products formed indicating the importance of the magnesium in PCR amplification while for magnesium dilution without touchdown (Figure 4), it was found that 2mM yielded the best results. Magnesium concentration is known to play a critical role in amplification as it can affect DNA strand denaturation, primer annealing specificity and enzyme fidelity. These observations are in agreement with earlier studies (Innis et al. 1990; Eeles et al. 1993). Even brief incubations of a PCR mix at temperatures significantly below theT_mcan result in primer-dimer formation and nonspecific priming. Hot-start PCR methods (Erlich et al. 1991;Ruano et al. 1992) can dramatically reduce these problems.

In this experiment, two methods were used to visualise the PCR products formed. They are, Polyacrylamide gel electrophoresis (PAGE) and Agarose gel electrophoresis. Agarose gel is the most popular medium for the separation of moderate and large-sized nucleic acids and have a wide range of separation but a low resolving power, since the bands formed in the gels tend to be indistinct and spread apart. This is a result of pore size and cannot be largely controlled (Stellwagen, 1998). However, Polyacrylamide gels are normally more difficult to prepare and handle, and it requires a longer time for preparation than agarose gels. However, polyacrylamide gels have a greater resolving power, can accommodate larger quantities of DNA without any significant loss in resolution and the DNA obtained from polyacrylamide gels is extremely pure (Guilliatt, 2002). Hence, they are better than agarose gels. It should be noted that polyacrylamide is a neurotoxin (when unpolymerized), but with proper laboratory care it is no more dangerous than various commonly used chemicals in the laboratory (Budowle & Allen, 1991).

Conclusion

Optimisation of Polymerase Chain Reaction is very important for PCR performance to minimize failures, avoid the production of non-specific products and increase specificity of the reaction. The knowledge of proper conditions allows to use PCR correctly and to receive good results.

According to the results of the research the most suitable annealing temperature was 62^oC, the most proper amount of concentration of magnesium was 2nMol, and the best template dilution was 2µl.

Those parameters give DNA amplification specificity and efficiency. (Harris and Jones, 1997).

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