Genetic engineering requires the combination of a number of different molecular genetics techniques. RFLP is a molecular method of genetic analysis that allows individuals to be identified based on unique nucleotide sequences found within a more highly conserved portion of DNA. Thus, the technique takes advantage of the polymorphisms in individual people's genetic codes. Execution of an RFLP analysis is dependant upon the following molecular "tools":
1. Restriction Enzymes
Perhaps one of the most important requirements for doing RFLP analysis is that we have restriction enzymes. Without them, there would be no way to cut DNA. The different restriction fragment lengths that arise from restriction enzyme cutting are what define the method.
2. Electrophoresis
In order to analyze the small DNA fragments that restriction enzymes produce, there has to be a method for separating them. Electrophoresis was made possible by the discovery that nucleotide fragments can be separated by moving them through a porous material (agarose) within an electric field and then visualized using fluorescent dyes or radionucleotides (as is the case for RFLP analysis).
3. Nucleotide Synthesis
DNA synthesis is essential for generating short fragments of DNA to make probes. In a probing technique called Southern Blotting, short pieces of DNA that complement certain fragments of the cut (restriction fragment) DNA, are synthesized using radiolabelled nucleotides. These "probes" bind to the DNA fragments after they are blotted onto a membrane. When X-ray film is exposed to the membrane, the radionucleotide probes create a pattern on the film which corresponds to the separation pattern of DNA on the gel.
We've had the ability to sequence DNA for many years. In fact, the first whole genome sequenced was in 1977. However, it has taken some improvements to the process, and automation (developed in the late 80s - early 90s) to make it fast and inexpensive enough for everyday use.
4. DNA Sequencing
The probes that are used in Southern Blotting must be synthesized using specific nucleotides, in the proper order, to make then complimentary to the separated fragments in an RFLP analysis. This is only possible because we are able to determine the sequence of a DNA strand. DNA sequencing methods typically require other molecular tools such as PCR and electrophoresis.