The construction of recombinant DNA molecules is a very sophisticated scientific technique involving the use of several key tools, one of which is restriction enzymes.
These enzymes are isolated from bacteria that recognise specific sequences in DNA.
Then, they are used to cut the DNA to produce restriction fragments. Restriction enzymes are assumed to provide a defence mechanism against several deadly viruses such as bacteriophages. They belong to a higher class of enzymes called nucleases which have the ability to cleave the phosphodiester bonds between the nucleotide subunits of nucleic acids.
The nucleases are categorised as exonucleases and endonucleases. Exonucleases remove nucleotides from the end of the polynucleotide chain. They hydrolyse phosphodiester bonds of terminal nucleotides while endonucleases cut the DNA at specific points and are thus known as ‘molecular scissors’. The second enzyme was called 'restriction endonuclease'. Thereafter, in 1968, scientists H.O. Smith, K. W. Wilcox and T. J. Kelley, while working on Haemophilus influenzae, isolated an enzyme called Hind II. Hind II was the first restriction endonuclease whose functioning depended on a specific DNA nucleotide sequence. It was observed that Hind II always cut DNA molecules at a particular point by recognising the position of the six base pairs. Therefore, the specific base sequence is also known as the recognition sequence for Hind II.
Restriction endonuclease plays a crucial role in the construction of the recombinant DNA molecule. It recognises a specific recognition sequence so it binds to the DNA and cuts the double-stranded DNA helix at specific points on its sugar phosphate backbones.
The specific recognition sequence actually identifies a palindrome in the DNA. Observe that the following sequence reads the same on the two DNA strands in five dash to three dash direction and vice versa. In a palindrome, the base sequence of the second half in the DNA strand represents the mirror image of the base sequence in the first half. Due to this, in the DNA double helix, the complementary strand also represents the same mirror image. Restriction endonucleases cut the DNA strand slightly away from the centre of the palindrome sites.
However, this cut is between the same two bases on the opposite DNA strands. As a result, single-stranded portions are left at the ends. Moreover, there are overhanging stretches called sticky ends on each strand. Such ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule together again. Thus, restriction endonucleases play a crucial role in the construction of recombinant DNA molecules, which have DNA from different sources. When the same restriction enzyme cuts different DNA molecules, all of them are left with similar sticky ends, which can be linked using DNA ligase. Therefore, restriction enzymes are very useful tools used by molecular biologists to manipulate DNA for scientific applications.