Optimization Of A Polymerase Chain Reaction Biology Essay

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Polymerase chain reaction is the process by which number of specific DNA sequence can be increased in vitro. PCR has become the central technique for scientific and clinical research. Millions of copies of short fragment of DNA are produced from this technique which includes the use of DNA polymerase and primers that are complementary to each strand. Each cycle of PCR is composed of three steps i.e. denaturation, annealing and extension. After every PCR thermal cycle, a new DNA segments are formed and these segments serve as a template for further amplification in the next thermal cycles. This laboration was performed with an aim to optimize different parameters including Taq polymerase, MgCl2, template DNA concentration and annealing temperature for different reaction conditions. PCR thermocycler was run according to the standard protocol and then obtained samples of different variables were run on the agarose gel with a ladder of 1 kb. The photograph of gel showed that Taq polymerase showed best results at 2U/ 20 µl while MgCl2 at 1mM, temperature at 70C and template DNA 5ng/ 20µl.

Introduction:

Polymerase chain reaction (PCR) is the process through which amplification of specific DNA sequence is achieved in vitro. (Glick and Pasternal, 1998). This process is simply the extension of DNA properties and is used in cloning experiments as well as in DNA manipulation experiments. (Reece, 2004). During 70s and 80s, there was development and invention of ways and means to use DNA polymerase properties as the production of complementary DNA, use of ligases and restriction enzymes to cut and then joining different segments together, a way for cloning. (Rathan and Aditi, 2010). The idea of polymerase chain reaction, stricked a chemist named Karry Mullis and he got the Nobel Prize for development of this procedure. (Reece, 2004).

The amplification of DNA sequences is achieved by a three step cycling process. The essential requirement of PCR involves, two synthetic oligonucleotide primers (usually 17 to 30 nucleotides in length) each is complementary to ends of different strands and produce 3'OH, a target DNA sequence that lies between pair of primers, a DNA polymerase that should be thermo stable and the four deoxyribonucleotides (dNTPs). (Glick and Pasternal, 1998).

The process of PCR is same as the cells undergo replication of their DNA. First of all, the double stranded DNA is unzipped and form two single strands of DNA. Then, DNA polymerase extends each strand from the primer till end. There are three steps involved in PCR which are repeated for 30 to 40 cycles. These three steps differ in their temperature requirement. The three steps of PCR are as follows.

Denaturation: A cycle comprising of heating and cooling is used for reversible denaturing of DNA double strand. In this step, a temperature 94C separates the target double stranded DNA molecule into its component strands.

Annealing: This step is carried out at a temperature of 45 to 60C in the presence of oligonucleotide primers. This temperature is dependent upon length and sequence of the primers used. In this step the oligonucleotide primers attach to their complementary sequences.

Extension: This step is carried out at a temperature of 72C. The DNA polymerase binds to the free 3' site of oligonucleotide primer and extends in 5' to 3' direction by using dNTPs. (Reece, 2004).

In each PCR cycle, there are three different steps which are temperature dependent. These steps are carried out in an automated heater which is fully programmed block and it contains reaction tubes. Generally, each cycle takes 3-5 min to complete. (Glick and Pasternal, 1998).

Earlier, the DNA polymerase used was not heat stable, so, on start of every cycle, it has to be again entered in the reaction. A thermophilic organism, thermas aquaticus that lived in hot springs contains DNA polymerase called Taq polymerase. Taq polymerase is thermostable and can tolerate temperature of 100C and can survive many PCR cycles without losing its activity. (Rathan and Aditi, 2010)

A copy of target DNA is produced at the end of first cycle which will act as template for the next cycle and in this way number of DNA copies increase exponentially. (Reece, 2004). The procedure for PCR is carried out in a simple way. First of all PCR machine is programmed to perform the desired the desire thermocycling and then the reaction tube containing the PCR mix are put in the block. Program is then run and the PCR product is analysed by running on the cast gels. (Rathan and Aditi, 2010)

In order to amplify a specific DNA sequence, there are several important factors that should be considered to achieve good results. These include choice of DNA polymerase, oligonucleotide primers, MgCl2 concentration, concentration of DNA template and temperature condition of the reaction. (Reece, 2004). In this lab, these important factors are manipulated to check their impact on overall PCR. The authors of this report were assigned to carry out PCR by optimizing four variable concentration of Taq polymerase 0.1, 0.5, 1 and 2 unit/20 µl of PCR mix and other reagents were same.

Material and Methods

Preparation of reaction mixture

For optimization of polymerase chain reaction the instruction are followed from the recombinant DNA technology laboratory compendium (Rathan and Aditi 2010). First of all we prepared the master mix from all PCR reagents except the variable reagent for four different PCR reactions. The variable reagent in our case was Taq DNA polymerase. Table 1 shows the formulation of master mix from stock solutions and concentration of

Taq polymerase for four different PCR reactions.

The Taq polymerase which used in this experiment was prepared by New England Biolabs. Inc.

Table 1. Master mix formulation

PCR reagents

Stock

concentration

Required concentration

Master Mix

Volume in PCR tubes with different Taq polymerase concentrations

Template DNA

5ng/μl

5ng/20μl

5 μl

1μl

1μl

1μl

1μl

dNTP*

10mM

200μM

2 μl

0.4μl

0.4μl

0.4μl

0.4μl

PCR buffer

10x

1X

10 μl

2μl

2μl

2μl

2μl

MgCl2

25mM

2mM

8 μl

1.6μl

1.6μl

1.6μl

1.6μl

Primer 1

10μM

0.5μM

5 μl

1μl

1μl

1μl

1μl

Primer 2

10μM

0.5μM

5 μl

1μl

1μl

1μl

1μl

Taq DNA polymerase

1U/μl

-----

-----

0.1μl

0.5μl

1μl

2μl

Deionised water

-----

-----

-----

12.9μl

12.5μl

12μl

11μl

Total reaction volume

35 μl

20μl

20μl

20μl

20μldNTP*=deoxyribonucleotide triphosphates

After preparing the four tubes with different variable Taq polymerase concentrations these tubes were transferred to PCR machine (thermocyler). The thermocycler was programmed according to protocol given in table 2.

Two primers were used. The forward primer was 5´- ATGACCATGGAGATGGCAAGCACCAGCTGCAA-3 and the reverse primer was 5´- GTGGTGCTCGAGCCAAGAAGGCTCAAAGAC-3´. The DNA template was 3061bp from the NALP3 gene.

Table 1. Steps of PCR.

Steps

Temperature(°C)

Time(min)

1

Initial denaturation

95ËšC

5 minutes

2

Denaturation

95ËšC

0.5 minutes

3

Annealing

59ËšC

0.5 minutes

4

Extension

72ËšC

1.5 minutes

Repeat steps 2,3and4 for 29 times

5

Final extension

72ËšC

8 minutes

6

Holding

4ËšC

Indefinite

The thermocycler works on three principles that are denaturation, annealing and extention of PCR product. The time duration in last step depends on length of final product. (Reece, 2004).

Preparation of agarose gel.

According to instructions given in Recombinant DNA technology laboratory compendium 0.8 % agarose gel was prepared. (Rathan and Aditi, 2010). Then the PCR product was loaded in the agarose gel and allowed it to run against 109mA electric field for 2 hours. The bands were compared with 1kb ladder and picture was taken at the end.

Results and Discussion

Different concentrations of Taq polymerase were used in this laboration i.e. 0.1 unit/20 µl, 0.5 unit/20 µl, 1 unit/20 µl and 2 unit/20 µl. After completion of PCR, the sample was then run on the agarose gel and different bands were obtained.

Figure 1: Gel image with variable Taq polymerase concentrations.

From figure 1, it is clear that out of four, three bands are found. The well containing 1U/20µl concentration of Taq polymerase did not show any band. It might be because of pipetting error. The well containing 2U/20µl show the optimum concentration for amplification in PCR. The little amplification was also observed with concentration of 0.5U and 0.1 from the figure 1.

Some groups use different concentration of MgCl2 i.e. 0mM, 1mM, 2mM, 3mM and 6mM. After completion of PCR, the sample was run on agarose gel and different bands were obtained.

Figure 2: Gel image for different MgCl2 concentrations.

From figure 2, it is clear that two bands are found out of five wells. The wells containing 0mM, 3mM and 6mM concentration of MgCl2 did not show any band. It might be because of pipetting error, mishandling of sample or not keeping the sample in ice properly. The well containing 1mM concentration of MgCl2 showed the optimum concentration for PCR. The band from the well containing 2mM concentration also showed some amplification.

Some other groups were assigned to check the optimization with different concentrations of template DNA. The concentrations used were 50ng/20µl, 10ng/20µl, 5ng/20µl, 1ng/20µl and 0.1ng/20µl.

Figure 3: Gel image for different template DNA concentrations.

From figure 3, it is clear that out of five, two bands are obtained. The wells with concentration of 50ng/20µl, 1ng/20µl and 0.1ng/20µl did not show any band. It might be because of pipetting error, mishandling of sample or not keeping the sample in ice properly. The well containing 5ng/20µl concentration of template DNA showed the optimum concentration for PCR. The band from the well containing 10ng/20µl concentration also showed some amplification.

Some other groups were assigned to check the optimization with different annealing temperatures. The different annealing temperatures used were 46.6 °C, 52.5 °C, 56 °C, 60 °C, 65 °C and 70 °C.

Figure 4: Gel image for different annealing temperatures.

From figure 4, it is clear that the well which was run with the annealing temperature 70°C showed the optimum temperature for PCR.

The optimum parameters found by the authors after observing the results of the whole group are summarized in the table

PCR parameters

Optimum

Taq polymerase

MgCl2

Template DNA

Annealing Temperature

2U/20µl

1mM

5ng/20 µl

70°C

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