Steps in DNA finger printing using RAPD method

 

 What is the full form of RAPD? How many primers are used in RAPD? Describe critically the various steps in DNA finger printing using RAPD method. Discuss the advantages and disadvantages of this method over other finger printing techniques. 

Full form of RAPD:-

       The term RAPD stands for Random Amplification of Polymorphic DNA. It was discovered by Williams et al. in 1991. It is a PCR based molecular marker technique but RAPD amplify segments of DNA which are essentially unknown to the workers, often PCR is used to amplify a known sequence of DNA.

Number of primers are used:-

      Only one random primer is used in the process of RAPD. Here a single short oligonucleotide primer is arbitrarily selected to amplify a set of DNA segment distributed randomly throught the genome. It is only 10 bases long and is usually obtained by in vitro DNA synthesis for which often a sophisticated equipment (called gene machine or oligonucleotide synthesizer) is used.

Steps in RAPD:-

       RAPD approach, save both cost and effort during DNA finger printing. The procedure of RAPD method is as follows-

(i) The genomic DNA of a selected strain or variety or species is isolated in a high molecular weight condition.

(ii) To this DNA, random primer (10bp) is added in excess amount.

(iii) The DNA and oligonucleotide mixture together with Taq DNA polymerase, buffer, dNTPs, are subjected to PCR cycle. In other words the mixture is subjected to repeated cycles of DNA denaturation and renaturation.

(iv) For denaturation, it is usually sufficient to heat the reaction mixture at 94°c for 30-60 seconds.

(v) During renaturation, following denaturation, the oligonucleotide will pair with the homologous sequence present at different location in the genomic DNA. This annealing of template and primer, occurs at 36°c for 2 minutes.

(vi) Therefore DNA polymerase will extend the olidnucleotide and copy the sequence continuous with the sequence with which the oligonucleotide had paired. This complementary strand synthesis occurs at 72°c for 1.5 minutes.

(vii) The repeated cycles of denaturation-renaturation- DNA replication in PCR will therefore amplify the sequence of genomic DNA. Amplification will take place only on two regions of the genome that have the sequence complementary to the random primer at both their 5' ends.

(viii) After several cycle of amplification, the DNAs are subjected to gel electrophoresis. The amplified DNA will form distinct bands which is usually detected by ethidium bromide staining and visible fluroscence under uv light.

Advantages:-

(i) It is a simple and easy technique. Need for a small amount of DNA (5-25 mg) makes it possible to work with populations which are inaccessible for RFLP analysis.

(ii) It involves non radioactive assays.

(iii) It needs a simple experimental set up requiring only a thermocycler and an agarose assembly.

(iv) It does not require species specific probne libraries, thus work can be conducted on a large variety of species where such probe libraries are not available.

(v) It provides a quick and efficient screening for DNA sequence based polymorphism at many loci.

(vi) It does not involve blotting or hybridization steps.

Disadvantages:-

(i) RAPD polymorphisms are inherited as dominant-recessive characters. This causes a loss of information relative to markers which show codominance.

(ii) RAPD primers are relatively short, a mismatch of even a single nucleotide can often prevent the primer from annealing, hence there is loss of band.

(iii) The production of non parental bands in the offspring of known pedigrees warrants its use with caution and extreme care.

(iv) RAPD is sensitive to changes in PCR conditions, resulting in changes to some of the amplified fragments.