The discovery of two types of enzymes in 1970s solved the problem of gene cloning. One is the restriction endonuclease and the other one is DNA ligase. The restriction endonuclease or restriction enzymes (RE) are synthesized by bacteria as a part of their defence mechanism.
These enzymes can cut DXA from any source by recognizing certain enzyme specific sequences on the DXA. The bacteria employ these enzymes to break down the harmful foreign genetic elements of other bacteria or bacteriophages. Bacteria also synthesise restriction modification enzymes that add methyl group to one or two bases within the specific recognition sequence of their own DXA; so that their own DXA are protected from restriction enzymes.
The first restriction endonuclease enzyme was isolated by D. Nathan and H. O. Smith (1970). There are three types of restriction enzymes: Type I, Type II and Type III. Type I and Type III are complex proteins having both endonuclease and methylase (modifying) activities.
They do not produce reproducible DXA fragments as they fail to cut the DXA inside the specific recognition sequences. Hence these two types of restriction enzymes are not used in recombinant DXA technology. Only Type II restriction enzyme is used and this is variously known as ‘molecular scalpcl”, “molecular Scissors”, “chemical scalpel” etc.
Most REs arc specific for short sequences, called palindromes and clcavc the phosphodicstcr bonds on both the DXA strands generating s’-OII and 5-P ends. A palindrome is a sequence of base pairs, which reads the same on both the strand if read in the same direction (from 5′- to 3′-). For example the sentence,”Madam I am Adam” is a palindrome as it reads the same in the reverse direction. Some REs cleave both the DXA strands asymmetrically around or on either side of the line of symmetry in the recognition sequence, yielding DXA fragments with sticky / cohesive ends.
Such type of cutting is known as staggered cut. A cohesive end has an overhanging single stranded fragment (either 3″-overhang or 5′-overhang), which is complementary to other cohesive end on other strand.
Such fragments having complementary cohesive ends circularize spontaneously and can again be linearised by heating. A few other Type II REs cleave both the strands symmetrically along the line of symmetry to produce DXA fragments with blunt ends / flush ends.
REs are named by three- alphabet abbreviation of the name of the bacterial species from which they are first isolated. The three alphabet abbreviation is followed by the strain of the bacterium and sometimes a Roman numeral is also used to designate multiple REs isolated from the same strain. In the three-alphabet abbreviation, the first one is for the bacterial genus and the second and third for bacterial
(I) Recognition sequences of three restriction endonuclease (Ilea III, PST I and Eco R I) along with line of symmetry (II) Precise sites of cleavage (Ill) a. blunt ends produced by I lac III b. PST I and c. Eco R I prducc cohesive ends.
For example, the first RE isolated from bacterium Escherichia coli strain RY 13 is named as Eco R I and the second RE from the same strain is Eco R II. Some of the restriction enzymes have the same recognition sequence and they are known as isoschizomers; but isoschizomers do not necessarily cleave at the same position. For example:
The other important enzyme in recombinant DNA technology is DNA ligase which joins two different DNA fragments. If the two fragments are generated by the same restriction enzyme, then they have complementary overhangs for which they anneal readily.
The ligase enzyme then forms phosphodiester bond between the two adjacent bases. Even DNA fragments with flush ends can also be joined by this enzyme. For this reason, this enzyme is also known as molecular glue. The most commonly used ligase is the T4 Ligase, which is purified from E. coli cell infected with T4 bacteriophage.
Several other enzymes are also used in recombinant DNA technology. Different DNA polymerases (I, II, III) are usede for DNA synthesis, repair and nick translation. Reverse transcriptase or RNA dependant DNA polymerase is used for reverse transcription of RNA to DNA. In reverse transcription, first a single stranded DNA is synthesized on a RNA template which is then used as a template for the synthesis of the second strand. Such a DNA reverse transcribed from RNA is known as cDNA (complementary DNA).