Random Amplified Polymorphic DNA (RAPD) Analysis | Molecular Biology

In this article we will discuss about the principle, requirements and procedure for Random Amplified Polymorphic DNA (RAPD) analysis.

Randomly amplified polymorphic DNA (RAPD) was developed by William et al. (1990) to detect genetic polymorphism in crop plants. Polymerase chain reaction (PCR) is an in vitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically amplified and modifications in its basic procedure has led to the develop­ment of RAPD for detecting variation at nu­cleotide level.

Principle:

RAPD reaction is based on the principle of polymerase chain reaction (PCR), an enzymatic assay for amplification of specific DNA segments in the target DNA.

Requirements:

1. RAPD random primers 5 p mol/1

2. dNTPs (dATP dGTP dCTP and

dTTP) 10 mM

3. MgCl₂ 15 mM

4. Template DNA 25 ng/µl

5. PCR buffer10x

(usually given with Taq polymerase).

6. Taq DNA polymerase. 5 U/µl

7. Agarose gel electrophoresis reagents.

8. Eppendorf tubes (1.5 and 0.5 ml).

9. PAGE or Agarose electrophoresis equip­ment.

10. Microcentrifuge.

11. Micropipettes (P₂, P₂₀, P₂₀₀, and P₁₀₀₀).

12. 0.2 ml thin walled PCR tubes.

13. Gel drier.

14. —20°C deep freezer.

15. Refrigerator.

16. Photo documentation system.

17. Laminar clean air flow hood.

18. U.V. transilluminator.

Procedure:

1. Add 2 µl of template DNA in PCR tubes, label and keep them on the rack.

2. Prepare reaction mixture in 1.5 ml Eppendorf tubes for required number of reactions. An addition of two tubes should also be prepared to compensate the pipetting loss.

3. For more than one primer reaction, mix­tures should be prepared separately.

Keep the tubes on ice while preparing PCR reaction mixtures and components should be added in the sequence indicated above.

4. Add 2 µl of reaction mixture to the 0.2 ml PCR tube with 2 µl of template DNA which makes the final volume to 25 µl. Mix well by patting the tube with fin­gers.

5. Centrifuge for few seconds to bring con­tents to the bottom of the tube (do not use vortex mixer, since it will inactivate enzyme).

6. Perform PCR reaction in a thermocycler as per programme given below:

7. Check the amplified product by running through 1.5% agarose gel stained with ethidium bromide.

8. Prepare 1.5% agarose gel with ethidium bromide.

9. Add 2 µl of 6x loading dye to 10 pi of PCR product and mix well.

10. Load the gel with PCR product along with a molecular weight marker (Lamda DNA/£coRI + Hind III double digest) and run the gel in 1x TBE at 50 V till the bromophenol dye front reaches the end of the gel.

10. View the gel using U.V. transillumina- tor. Document the profile using a gel documentation system for further analy­sis.