BY: RAHUL ANDHARIA (MSIWM001)
Molecular marker is a sequence of DNA in the genome that can be traced and identified. RAPD is a molecular marker that helps to identify genetic variations. Single arbitrary primer is used in RAPD.
It is used to identify nucleotide polymorphism. This method came into existence during the year 1990 when William et.al first used this approach.
- Shorter oligonucleotide primers binding to different loci is used for amplification of random sequences from complex DNA template.
- The amplified PCR product depends on length and size of primer and the target genome.
- RAPD is a PCR based method. All the components of PCR are required to perform RAPD like DNA template, dNTPs, reaction buffer, Mgcl2(cofactor), arbitrary primer, Taq polymerase.
- First step basically involves denaturation, where the PCR products are kept at higher temperature of 94 degree Celsius.
- Temperature is lowered to 40-65 degree Celsius in annealing process where the primer binds to the target DNA sequence.
- Further Taq polymerase adds DNA-hybrid primers to 3’ end and extends to the other end of target sequence.
- The process is repeated until complete replication of amplified product is achieved.
Outline of RAPD Analysis:
- Genomic DNA is isolated.
- The isolated DNA is denatured.
- DNA template is annealed with primers- Annealing.
- Complementary strands of DNA are synthesized after the extension phase.
- Gel electrophoresis is used to identify amplified products.
Procedure of RAPD:
Materials Required: PCR components, RAPD arbitrary primers, gel electrophoresis reagents and equipments, Eppendorf’s, PCR tubes, micro centrifuge, micropipettes, deep-freezer, UV transilluminator connected with a system.
- Master mix is prepared(control and sample) with all the PCR components.(thaw(keep it in ice) and vortex).
- The master mix is aliquoted in PCR tubes.
- DNA templates is added in the PCR tubes.
- The tubes are placed in allotted blocks in the PCR thermocycler machine.
- The reaction setting is adjusted and different temperatures are set for different cycles.
- Ideal reaction is: step 1 for 94 degree Celsius (1min), step 2 for 35 degree Celsius (1min) and step 3 at 72degree Celsius for 5min. For final step, temperature is again 72 degree Celsius for 5min.
- DNA loading dye is added to the amplified product and Agarose gel electrophoresis is performed. (% of agarose gel is determined based on sample size).
- The gel is visualised under UV trans illuminator connected to a system.
Applications of RAPD:
- As molecular markers in plants: Using RAPD, genetic maps are constructed in plants. For example- In coffee 15 linkage groups are constructed using RAPD marker.
- Desirable trait can be selected directly using RAPD. This marker can be screened with the desired trait anytime during breeding programs. This gives breeders advantage, as they can trace the desired trait.
- RAPD is widely used to identify certain genes which are resistant to diseases. For example- In barely crop, the gene rp94 is resistant to stem rust.
- Polymorphism studies can be done using RAPD.
- RAPD markers are used in evolutionary and population genetics for identifying genetic variations among different species.
- Used in germplasm(genetic material of Germ cells) characterization.
- RAPD can be employed to detect somaclonal variations, variations observed in somatic cells of regenerated plants.
RAPD PCR is used for detection of genetic polymorphism in leishmania strains(parasites causing leishmaniasis). Example- This type of test was successfully performed in Tunisian patients.
Merits of RAPD:
- For designing specific primers, no DNA probes and specific sequences is required.
- It is a quick, simple and efficient method as it does not involve blotting or hybridization steps.
- Amount of DNA required in the process is small compared to other methods.
- Number of fragments are higher.
- Compared to other assays, market cost for RAPD is relatively low.
Demerits of RAPD:
- Since RAPD markers are Dominant, it will be difficult to distinguish whether a particular DNA sequence is amplified from heterozygous locus or homozygous locus.
- RAPD is exclusively laboratory based technique and hence requires all the necessary components and suitable PCR conditions to obtain desired result.
- This method is sensitive to changes in DNA, PCR components and PCR conditions and hence has problems with reproducibility.
- Null alleles ( it is a non-functional allele caused due to mutations) cannot be detected directly by this method.