RFLP analysis is one of the older methods of DNA testing.
First developed in the 1970s
RFLP stands for "restriction fragment length polymorphism.
The first two words refer to the process of DNA fragmenting with specially applied enzymes.
Once this takes place, the DNA strands break up into various lengths.
Polymorphism in Greek means "many shapes."
This word references the multiple shapes or lengths, that can result in a definitive match distinctive to each human being.
RESTRICTION FRAGMENT LENTH POLYMORPISM
A restriction enzyme cuts the DNA molecules at every occurrence of a particular sequence, called restriction site.
For example, HindII enzyme cuts at GTGCAC or GTTAAC.
If we apply a restriction enzyme on DNA, it is cut at every occurrence of the restriction site into a million restriction fragments each a few thousands nucleotides long.
Any mutation of a single nucleotide may destroy or create the site (CTGCAC or CTTAAC for HindII) and alter the length of the corresponding fragment.
RFLP analysis is the detection of the change in the length of the restriction fragments.
RFLP analysis may be subdivided into single- (SLP) and multi-locus probe (MLP) paradigms.
Usually, the SLP method is preferred over MLP because it is more sensitive, easier to interpret and capable of analyzing mixed-DNA samples.
HOW RFLP ANALYSIS WORKS?
The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digestion.
The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis.
Then transferred to a membrane via the Southern blot procedure
The DNA is harvested from analyzed samples
A "restriction enzyme," applied to each complete DNA molecule will create fragments of various lengths by dissolving the DNA.
An electric current is applied on the Gel
DNA is negatively charge
DNA fragments will start moving towards the
positively charged side
Smaller fragments move faster
After some time, we have a separation of the
different fragment lengths
DNA FRAGMENTS MOVE
The colored solution provides an indication to how much the DNA has traveled on the Gel.
After the electropheresis is completed, the DNA fragments in the gel can be stained with a blue dye or made radioactive to make them more visible.
If the molecules fluoresce under a ultravoilet light, a photograph or an autoradiogram can be taken.
APPLICATIONS OF RFLP
In DNA analysis RFLP has many uses, including:
Restriction fragment length polymorphism (RFLP) is most suited to studies at the intraspecific level or among closely related taxa. Presence and absence of fragments resulting from changes in recognition sites are used identifying species or populations.