MOLECULAR BIOLOGY AND GENETIC ENGINEERING

Aim of the Experiment: Polymerase Chain Reaction and analysis by agarose gel electrophoresis.

Introduction:

Polymerase Chain Reaction (PCR) is an in vitro method of enzymatic synthesis of specific DNA fragment developed by Kary Mullis in 1983. It is a very simple technique for characterizing, analyzing and synthesizing DNA from virtually any living organism (plant, animal, virus, bacteria). PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material called as template DNA.

A basic PCR requires the following components:

1. DNA template that contains the region to be amplified

2. Two primers complementary to the 3’ ends of each of the sense and anti-sense strand of the DNA

3. Thermostable DNA polymerase like Taq, Vent, Pfu etc.

4. Deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP), the building blocks from which the DNA polymerase synthesizes a new DNA strand.

5. Buffer solution which provides a suitable chemical environment for optimal activity and stability of DNA polymerase.

6. Bivalent magnesium/manganese ions, which are necessary for maximum Taq polymerase activity and influences the efficiency of primer to template annealing. Thermal cycler to maintain constant reaction temperature throughout the cycles.

Principle:

The purpose of a PCR is to amplify a specific DNA or RNA fragment. PCR comprises of three basic steps:

1. Initialization step: This step consists of heating the reaction mixture to 94–96oC for 1–9 minutes to initiate breaking of the hydrogen bonds in DNA strands.

2. Denaturation step: This step is the first regular cycling event and consists of heating the reaction mixture to 94–98o C for 20–30 seconds. As a result the template DNA denatures due to disruption of the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

3. Annealing step: In this step the reaction temperature is lowered to 50–65o C for 20–40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is 3-5o C below the Tm (melting temperature) of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

4. Extension/Elongation step: In this step, the temperature depends on the DNA polymerase used. Taq polymerase has its optimum activity at 75–80o C. Commonly a temperature of 68-72o C is used with this enzyme. The DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by incorporating dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both upon the DNA polymerase used and on the length of the DNA fragment to be amplified. The DNA polymerase will polymerize a thousand bases per minute at its optimum temperature. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential amplification of the specific DNA fragment.

5. Final elongation: This single step is occasionally performed at a temperature of 70–74o C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended. Denaturation, annealing and extension steps are repeated 20-30 times in an automated thermocycler that can heat and cool the reaction mixture in tubes within a very short time. This results in exponential accumulation of specific DNA fragments, ends of which are defined by 5’ ends of the primers. The doubling of the number of DNA strands corresponding to the target sequences allows us to estimate the amplification associated with each cycle using the formula; Amplification = 2n , where n = No. of cycles.

6. Final hold: This step may be employed for short-term storage of the reaction mixture at 4°C for an indefinite time.

Materials required

Glasswares: Measuring cylinder, Beaker

Reagents: Ethidium bromide (10 mg/ml), Distilled water

Other requirements: Thermocycler, Electrophoresis apparatus, UV Transilluminator, Vortex Mixer, Micropipettes, Tips, Adhesive tape, Microwave/ Hotplate/ Burner, Crushed ice.

Procedure

1. Preparation of master mix for PCR: To a PCR tube add all the following ingredients in order.

2. Follow the below given fig 3.

3. Tap the tube for 1–2 seconds to mix the contents thoroughly.

4. Add 25 μl of mineral oil in the tube to avoid evaporation of the contents.

5. Place the tube in the thermocycler block and set the program to get DNA amplification.

NOTE: It is not essential to add mineral oil if the thermocycler is equipped with a heating lid.

PCR Amplification Cycle: Carry out the amplification in a thermocycler for 30 cycles using the following reaction conditions.

Agarose Gel Electrophoresis:

1. Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use.

2. Preparation of agarose gel: To prepare 50 ml of 0.8% agarose gel, add 0.4 g agarose to 50ml of 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave or hot plate by swirling the glass beaker/flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow the solution to cool down to about 55-60o C. Add 0.5μl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

3. Loading of the DNA samples: Load 3 μl of ready to use DNA ladder into the first well. Add 2 μl of 6X Gel loading buffer to 10 μl of PCR product. Load the PCR samples into the following wells.

Note: Care should be taken while pipetting out the PCR product from the tube so as to avoid the mineral oil layer.

4. Electrophoresis: Connect power cord to the electrophoretic power supply according to the conventions: Red Anode and Black-Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

Observation and Result:

After completion of the PCR, perform agarose gel electrophoresis. Compare the amplified product with the ladder and determine its size.

Interpretation:

After performing agarose gel electrophoresis, one can check the amplification of a specific PCR product. The optimized conditions result in the amplified PCR product of desired size.

Reference Material:

https://himedialabs.com/TD/HTBM016.pdf.

Techniques in Biochemistry and Molecular Biology

Cloning - T.A. Brown.

Questions

1. If you observe non-specific/spurious bands, what are the possible reasons and solution?

2. What if you get poor or no amplification of bands under gel? justify giving solutions.

3. What is the possible reason for primer-dimer problem?

Developed by

Dr. Saroj Shekhawat,

Assistant Professor, Biotechnology

saroj.shekhawat@gsfcuniversity.ac.in

Ms. Bhargavi Sonavane,

Teaching Assistant, Biotechnology,

bhargavi.sonavane@gsfcuniversity.ac.in