Polymerase Chain Reaction

BACKGROUND

Polymerase Chain Reaction

The amplification of DNA in defined regions by polymerase chain reaction (PCR) is very important in molecular biology. The use of PCR is described in chapter 12 of your textbook. You will be amplifying a small region of DNA (the mouse hmga2 gene, ~330 bp) using the two primers below.

In your laboratory report you should discuss what the primers do, how they work, and why it is important to use a thermally stable polymerase such as Taq.¹

Aim of the experiment is to amplify a DNA sample by the use of PCR.

REQUIREMENTS

10 x PCR buffer containing Mg²⁺

Template DNA pMGM1

Primers: 5′-CATATGAGCGCACGCGGTGAGGG-3′ forward primer 5′-CTCGAGCTAATCCTCCTCTGCGGA-3′ reverse primer; dNTP; Taq DNA polymerase^4,6

PROCEDURE

The PCR reaction is usually set up in a thin walled microcentrifuge tube. The thin wall allows the transfer of heat from the heat block (thermal cycler) to the liquid inside to occur rapidly.

The PCR reaction contains each of the four deoxynucleoside triphosphates (dNTPs: dGTP, dATP, dTTP, dCTP), MgCl2 (required by the DNA polymerase; usually 2 mM), the DNA polymerase (Taq), template DNA (the DNA that is being amplified), primers (define the region to be ampli- fied), and a buffer.

The DNA polymerase Taq comes from a thermophile, an organism that likes hot temperatures, even warmer than those in Florida. To set up a 50 µl PCR reaction following components are required, 5 µl 10 x PCR buffer;³

4 µl 25 mM MgCl2

1 µl template DNA (10 ng)

1 µl dNTP (10 nmol)

1 µl forward primer (20 pmol)

1 µl reverse primer (20 pmol)

0.5 µl Taq DNA polymerase

36.5 µl H2O

After preparation of PCR reaction mixture, following steps have to be followed to amplify the DNA in the thermal cycler of PCR instrument; Firstly, the reaction starts with the separation of two strands of DNA which is called as denaturation step at 96^°C, for 15 seconds.^1,5

Then, as the temperature starts to cool down i.e; at 55^°C, the two separated strands start to renature with the base pairs at the complementary regions and this step is also known as annealing which lasts foe 30 seconds.

The last step is of synthesis of new strands which occurs at 3ˊ-hydroxl end of each primer and the temperature has to be maintained at this step as DNA polymerase is only responsible for polymerization of DNA of which Taq DNA pol is having optimum temperature i.e; around 75^°C and the reaction canbe stopped by raising the temperature to 95^°C.^2,3

As PCR will run 18 to 20 cycles and each cycle lasts upto 3-5mins therefore the final extension is performed at 72^°C for 5 min.

(The thermal cycler rapidly changes temperature so that the DNA alternately denatures, anneals, and then extends. This cycle of denaturation, annealing, and extension is repeated 18 to 20 times).

For further analysis of the sample i.e; the resulting PCR reaction mixture has to be run in a 6% polyacryamide gel. Polyacrylamide gels are used to analyze shorter pieces of DNA and have been used to sequence DNA.

Polyacrylamide gels are made by polymerizing acrylamide with the crosslinking agent bisacrylamide. Acrylamide purchased for molecular biological application, normally already contains the bisacrylamide, although in this case it is need to be added. The polymerization reaction is a radical reaction initiated by ammonium persulfate and TEMED.

Note:

Acrylamide and bisacrylamide are toxic and Polyacrylamide is much more benign.

The need of the electrophoresis is to prepare 50 mL of a 30% acrylamide/bis-acrylamide solution in water with a weight ratio of 29 parts acrylamide : 1 part bisacrylamide. Once everything has dissolved filter the solution and then store it at 4°C in a brown glass bottle.

The 10% Ammonium Persulfate (APS) solution has to be prepared freshly. The stock solutions has to be pepared using 30% acrylamide, 10% Ammonium Persulfate (APS), 5 x TBE and TEMED; 60 ml of 6% polyacryamide gel, then from the stock the gel is prepared by mixing 12 ml of 30% acrylamide in 35.5 ml of water and mixed properly, then 12 ml of 5 x TBE and 480 µl of freshly prepared 10% APS to which 20 µl of TEMED is added.²

Then the gel caster was first assembled, and then 6% gel mix was added to gel caster. After that add the comb to the gel, and wait until gel is fully polymerized (about 30 to 60 min). The gel can then be loaded and the running buffer is 1 x TBE.

The gels are then visualized using ethidium bromide and UV light.

CONCLUSION

From the above experiment by observing the number of bands in the gel, it can be concluded that the sample has been amplified after running in thermal cycler of PCR.

REFERENCES

1. www.creativebiomart.net/blog/principle-and-procedure. Accessed 20 August 2017.
2. Biochemistry lab manual by Fenfei Leng Florida International University Department of Chemistry Revised, spring; 2009: 27-29.
3. Satayanarayan U. Biotechnology; 2010: 109-112.
4. Ninfa J, Alexander P, Ballou D. Fundamental laboratory approaches for biochemistry and biotechnology., Wiley; 2004.
5. Sambrook J, Russel DW. Molecular cloning: A laboratory Manual 3^rd Ed. Cold spring harbor lab press; 2001.
6. Janarthanan S, Vincent S. Practical biotechnology -Methods and Protocols.
   

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