Restriction Maps and RFLPS | Genetic Engineering

In this article we will discuss about the restriction maps and RFLPS. Also learn about the significance of restriction maps.

The number of cuts that a restriction enzyme makes in a segment of double-stranded DNA depends on the size of that DNA, its sequence, and the number of base pairs in the recognition sequence of the particular enzyme.

That is, a restriction enzyme with only three base pairs in its recognition sequence will cut more times than with six base pairs in its sequence, since the probability of a sequence occurring by chance is a function of the length of that sequence.

A sequence of three bases occurs more often by chance (1/4³ = 1/64 base pairs) than a sequence of six bases (1/4⁶ = 1/ 4096 base pairs). Hind II, for example, cuts the circular DNA of tumour virus SV40 into eleven pieces; some restriction enzymes can cut E. coli DNA into hundreds of pieces. The product of the action of a restriction enzyme on a DNA sample is called a restriction digest.

Using electrophoresis, one can separate the fragments of a restriction digest by size. With a technique, we can locate the restriction sites on the original gene on piece of DNA. That is, we can construct a map, called restriction map, of the restriction recognition sites that will give us the physical distance between sites, in base pairs.

This restriction map is extremely valuable for several reasons. For example, when the radioactive nucleotide tritiated thymidine was added for a very short period of time during the beginning of the DNA replication in SV40 viruses, the radioactivity always reappeared in only one restriction fragment. The experiment demonstrated that SV40 replication started from a single unique point; that point was localized to a particular segment of the SV40 chromosome.

Further, a restriction map often allows researchers to correlate the genetic map and the physical map of a chromosome. Certain physical changes in the DNA, such as deletions, insertions, or nucleotide changes at restriction sites, can be localized on the genetic map.

These changes can be seen as changes in size, or in the total absence, of certain restriction fragments when compared with wild-type DNA. This information allows us to see changes in the DNA; is also gives us information about the evolution of species.

The difference in fragment sizes are called restriction fragment length polymorphisms (RFLPs) and have proved valuable in pinpointing the exact location of genes and determining the identity or relatedness of individuals. A restriction digest is also useful for isolating short segments of DNA that can be easily sequenced.

Significance of RFLPs:

Restriction fragment length polymorphism (RFLPs) obtained from restriction digests, are proving to be very valuable genetic markers in two areas of study: human gene mapping and forensics (i.e., DNA fingerprinting).

In a restriction digest of the whole human genome, there might be thousands of fragments from a single restriction enzyme. Unique probes have been developed for southern blotting these digests. Genetic variation usually comes in the form of a second allele that, due to a mutation, lacks a restriction site and is therefore part of a larger piece of DNA.

Some probes have uncovered hyper-variable loci with many alleles (in fact, any person has only two of the many possible alleles). A population’s genetic variation is generated because these hyper-variable loci contain many tandem repeats of short (10 to 60 bp) segments.

Due to unequal crossing over, just one of these loci, called variable-number-of-tandem repeats (VNTR) loci, can generate much variation. As a result, probing for one of these VNTR loci in a population reveals many alleles.

The southern blots of such restriction endonuclease digests create a DNA fingerprint of extreme value in forensics. DNA extracted from blood or semen samples left by a criminal can be compared with DNA patterns of suspects.

When a single probe recognizes a number of different- loci, each individual will have many bands on a southern blot, with most people producing unique patterns. In one system, developed by Alec Jeffreys, a single probe locates fifty or more variable bands per person.

If Jeffreys’s probes are used to compare the patterns, the chance that the two patterns would match randomly is generally small. This technique, thus, has greater power to identify individuals than using the prints from their fingertips.