Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms can be differentiated on the basis of analysis of patterns derived from processing of their DNA. Polymorphisms are inherited differences found among the individuals in more than 1% of normal population.
If two organisms differ in the distance between sites of cleavage which are called the restriction sites of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA and will be digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species and even strains from one another. Restriction endonucleases are the enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites which are usually 4 to 6 base pairs in length.
There are various applications of RFLPs. Some of them are listed below
- RFLPs are used in paternity identification or criminal cases to determine the belonging of the DNA sample.
- RFLPs can be used to determine the disease prognosis of an individual.
- RFLPs can be used to measure recombination rates which can lead to a genetic map with the distance between RFLP loci measured in CentiMorgans.
- Also used for fingerprinting.
RFLP refers to a variation in a sequence of DNA that has a restriction site on each end with a target sequence in between. A target sequences that segment of DNA that binds to a probe by attaching to the complementary base pairs. A probe is a sequence of single-stranded DNA that has been tagged with radioactivity or an enzyme so that the probe can be detected. When probe base pairs to its target, the investigator can detect this binding and know where the target sequence is, as the probe is detectable. SSR and RFLP molecular markers are co-dominant in nature. When the southern blot is performed the RFLP produces a series of bands with a specific combination of restriction endonucleases and probe sequences. RFLP can be used for fingerprinting.
RAPD (Randomly Amplified Polymorphic DNA)
RAPD technique is based on PCR and does not utilize restriction enzymes as in case of RFLP. So this technique is both cost effective and easy as compared to RFLP. The strains that have a segment of DNA having both the ends which are homologous to the oligonucleotides used, a distinct band will be seen on the agarose gel and this band will be absent in strains in which both ends of this sequence of genomic DNA is either modified or deleted. While the RAPD method is empirical, its simplicity of use and the eventual identification of some stretches of DNA make it a popular means of identifying breeds. In this technique, random sequence is used as primers for PCR amplification of DNA from different strains. Only one (not pair) primer is used per PCR reaction. Short primers (oligonucleotides) about 10-12 mers are used.
The procedure for obtaining RAPD involves following steps:
- Isolation of genomic DNA from selected strain.
- Addition of selected oligonucleotides which serve as a primer and is obtained by in vitro DNA synthesis.
- PCR amplification of mixture in which oligonucleotides will pair with homologous sequences present at different locations in the genomic DNA.
- Gel electrophoresis of amplified product. Amplified product will be those regions of the genome that have the sequence complementary to the random primer at both their ends.
Amplified Fragment Length Polymorphism (AFLP) is a selective PCR based amplification technique in which amplification of restriction fragments from a total digest of genomic DNA. This method is capable of detecting polymorphism in DNA. The method was discovered by Vos and Zabeau in 1993. AFLP technique is used for identification of genetic variation in closely related species, for criminal and paternity testing (finger printing), to generate QTL maps etc. AFLP and RAPD molecular markers are dominant in nature. This technique uses restriction enzymes to digest genomic DNA which is followed by ligation of adaptors to the sticky ends of restriction fragments.
The procedure of AFLP involved following steps:
- In the very first step the digestion of total genomic DNA with restriction enzymes is performed along with the annealing of restriction half site specific adaptors to restriction products.
- Second step involves specific amplification of the restriction fragments with two PCR primers that have adaptor and restriction site specific sequences.
- Final step involves Electrophoretic separation of amplified products on a gel matrix and visualization of the band pattern.
SSRs Simple Sequence Repeats (SSR), also known as microsatalites, usually consist of di ot tri-nucleotide repeats (ATGATGATG) is an example of a tri-nucleotide repeat (ATG)). They are the small segments of DNA usually 2-5 base pair that repeats itself number of times. They are widely used for DNA profiling in kinship analysis and in forensic identification. These sequences are used in fingerprinting and also help in detection of allelic variations.