Restriction fragment length polymorphism (RFLP):

The RFLP is considered to be more accurate than the PCR, mainly because the size of the sample used more, use of a fresh DNA sample, and no amplification contamination. The RFLP, however, require longer time period to complete the analysis and is costly. The first step in this process is to isolate the DNA from the sample material to be tested. As mentioned, the sample size for RFLP test must be large enough to get the proper result. Once the required size of the sample is available , the DNA is isolated from the sample and is subjected to restriction digestion using restriction enzymes. The digested DNA sample is then separated by agarose gel electrophoresis, in which the DNA is separated based on the size. The next step is transfer of separated DNA from gel slab onto the nitrocellulose membrane to hybridize with a labeled probe that is specific for one VNTR region (radio activity labeled complimentary sequence for VNTR region nucleotide sequence). This technique of transferring and hybridizing DNA onto nitrocellulose membrane is known as southern blotting, a most widely used DNA detection technique by molecular biologists. After the hybridization with the radioactive probes, the X- ray film is developed form the southern blotting and only the areas where the radioactive probe binds will show up on the film. Now these bands when compared with the other known samples, will give the final result of the DNA Fingerprinting.