Rflp
Restriction Fragment Length Polymorphism (RFLP)
Restriction fragment length polymorphism (RFLP) analysis was one of the first techniques to be widely used for detecting variation at the DNA sequence level. The principle behind the technology rests on the possibility of comparing band profiles generated after restriction enzyme digestion in DNA molecules of different individuals. Diverse mutations that might have occurred affect DNA molecules in different ways, producing fragments of variable lengths. These differences in fragment lengths can be seen after gel electrophoresis, hybridisation and visualisation.
Isolating DNA
Isolating DNA is the first step for many DNA-based technologies. DNA is found either in nuclear chromosomes or in organelles (mitochondria and chloroplasts). To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane, and so appropriately separate the DNA from other cell components. When doing so, care must be taken to ensure the process does not damage the DNA molecule and that it is recovered in the form of a long thread.
Complications in this step includes Breakage during isolation, DNA degraded by nucleases, joint isolation of polysaccharides, isolation of secondary plant metabolites.
Restriction digestion and gel electrophoresis
Extracted DNA is digested with specific, carefully chosen, restriction enzymes. Each restriction enzyme, under appropriate conditions, will recognise and cut DNA in a predictable way, resulting in a reproducible set of DNA fragments (‘restriction fragments’) of different lengths. The millions of restriction fragments produced are commonly separated by electrophoresis on agarose gels. Because the fragments would be seen as a continuous ‘smear’ if stained with ethidium bromide, staining alone cannot detect the polymorphisms. Hybridisation must therefore be used to detect specific fragments.
DNA transfer by Southern blotting
In this method, the gel
Restriction fragment length polymorphism (RFLP) analysis was one of the first techniques to be widely used for detecting variation at the DNA sequence level. The principle behind the technology rests on the possibility of comparing band profiles generated after restriction enzyme digestion in DNA molecules of different individuals. Diverse mutations that might have occurred affect DNA molecules in different ways, producing fragments of variable lengths. These differences in fragment lengths can be seen after gel electrophoresis, hybridisation and visualisation.
Isolating DNA
Isolating DNA is the first step for many DNA-based technologies. DNA is found either in nuclear chromosomes or in organelles (mitochondria and chloroplasts). To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane, and so appropriately separate the DNA from other cell components. When doing so, care must be taken to ensure the process does not damage the DNA molecule and that it is recovered in the form of a long thread.
Complications in this step includes Breakage during isolation, DNA degraded by nucleases, joint isolation of polysaccharides, isolation of secondary plant metabolites.
Restriction digestion and gel electrophoresis
Extracted DNA is digested with specific, carefully chosen, restriction enzymes. Each restriction enzyme, under appropriate conditions, will recognise and cut DNA in a predictable way, resulting in a reproducible set of DNA fragments (‘restriction fragments’) of different lengths. The millions of restriction fragments produced are commonly separated by electrophoresis on agarose gels. Because the fragments would be seen as a continuous ‘smear’ if stained with ethidium bromide, staining alone cannot detect the polymorphisms. Hybridisation must therefore be used to detect specific fragments.
DNA transfer by Southern blotting
In this method, the gel