Question

Explain the principles of PCR using the following
headings .

DNA isolation

Amplification

Gel electrophoresis of PCR products

Answer 1

The polymerase chain reaction(PCR) is an in vitro DNA synthesis method during which DNA is amplified using Taq DNA polymerase.
The PCR- polymerase chain reaction is a temperature-dependent process of DNA amplification. The machine used in the PCR technique is known as a Thermocycler.

DNA isolation: first step before carrying out PCR is DNA isolation which we want to amplify, this can be achieved by various methods like, organic extraction that is phenol chloroform method, non organic method like salting out and proteinase K treatment and adsorption method silica gel membrane.
Organic extraction method most widely used:
First step is Cell lysis can be done using nonionic detergent (sodium dodecyl sulfate), Tris–Cl, and Ethylene diamine tetraacetic acid (EDTA), this step is followed by removal of cell debris by centrifugation. Protease treatment is then used to denature proteins. Organic solvents such as chloroform, phenol, or a mixture of phenol and chloroform (phenol/chloroform/isoamyl alcohol ratio is 25:24:1) can be used for denaturation and precipitation of proteins from nucleic acid solution, and denatured proteins are removed by centrifugation and wash steps. RNAse treatment is done for the removal of unwanted RNA. Precipitation with ice-cold ethanol is performed for concentrating DNA. Nucleic acid precipitate is formed, when there is moderate concentration of monovalent cations (salt). This precipitate can be recovered by centrifugation and is redissolved in TE buffer or double-distilled water further Agarose gel electrophoresis for confirmation of genomic DNA isolation. Further isolated DNA can be amplified by PCR method.
Nowadays , kit based methods for isolation of DNA are also widely used.

Amplification:
The principle of the PCR is generally based on the temperature variations of heating and cooling- thermocycling reaction divided into three steps:
Denaturation- The dsDNA becomes single-stranded at a higher temperature during denaturation. Here hydrogen bonds between two DNA strands break.In a denaturation two single-stranded DNA forms from the double-stranded one. At 94ºC temperature, the double-stranded DNA opens up by breaking hydrogen bonds. The process of denaturation is followed by the initial denaturation for 5 to 7 minutes at the same temperature.

Annealing- in The primer binds or anneals to its exact complementary sequence on a DNA during the annealing step. The primer provides a site for the initiation of synthesis. After the denaturation, primer anneals to ssDNA at its exact annealing temperature. Base on the GC content of primers, every primer has its own annealing temperature. The annealing temperature is ranging from 55ºC to 65ºC. Annealing temperature lower than that leads to non-specific bindings while higher temperature leads to amplification failure. 45 seconds to 1 minute enough for the second step, annealing for more than 1 minute causes non-specific amplification.

Extension- Taq DNA polymerase uses the 3’ end of the primer and it starts DNA synthesis by adding nucleotides to the growing DNA strand.
After the binding of the primer, its time to expand the DNA strand. Here in extension step the Taq DNA polymerase comes in action and adds dNTPs to the DNA strand. The temperature for the extension is 72ºC for 45 seconds.
After completing all steps one more time the final extension is performed for 7 minutes.

All three steps are repeated for 30- 35 cycles and in each cycle the DNA becomes double.
1. Initial denaturation is carried out at 90°C - 95°C for 5- 7 minutes
2. Denaturation is carried out at 90°C -95°C for 45 sec.
3. Annealing is carried out at 56°C -65°C for 45sec
5. Extension is carried out at 72°C for 45 sec
6. Final extension is carried out at 72°C for 5-7 minutes.
Component. Volume (microlitre)
1.template. 1
2. Buffer. 5
3. Primer I 2
4.primer II 2
4dNTPs. 1
5.Enzyme mixture 0.75
6.water. 38.25

Once PCR is carried out PCR product which are formed are analysed most commonly by gel electrophoresis method.It is routinely used for analysis of PCR products,
Gel electrophoresis of PCR product:

The negatively charged DNA molecules migrate towards the positive charge under the influence of constant current, thus the separation depends on the mass and charge of DNA. The DNA molecules are forced to move through the agarose gel pores.
1.Pouring a Standard 1% Agarose Gel:Measure 1 g of agarose
2.Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
3.Microwave for 1-3 min until the agarose is completely dissolved
4.Let agarose solution cool down to about 50 °C
5.ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
6.Pour the agarose into a gel tray with the well comb in place.
7.Add loading buffer to each of your DNA samples.
8.Once solidified, place the agarose gel into the gel box (electrophoresis unit).
Fill gel box with 1xTAE (or TBE) until the gel is covered.
9.Carefully load a molecular weight ladder into the first lane of the gel.
10.Carefully load your samples into the additional wells of the gel.
11. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
12.Off thepower, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.If you did not add EtBr to the gel and buffer, place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, replace EtBr solution with water and destain for 5 mins.

Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel. By referring to ladder one can infer the size of PCR product.