Unless the vector and source DNA are cut, fragments seperated and joined, the desired recombinant vector molecule cannot be created.
(a) How are the desirable DNA sequences cut ?
(b) Explain the technique used to seperate the cut fragments.
(c) How are the resultant fragments joined to the vector DNA molecule ? (5 marks)
(i) DNA sequences of the vector as well as the source are cut by the same restriction enzyme like
EcoRI, in a palindromic sequence.
(ii) East restriction endonuclease functions by 'inspecting' the length of a DNA sequence. Once it
finds its specific recognition sequence, it will bind to the DNA and cut each of the two strands of
the double helix at specific points in their sugar-phosphate backbones. Each restriction endonu-
clease recognises a specific palindromic nucleotide sequence in the DNA.
Restriction enzymes cut the strand of DNA between the same two bases on the opposite
strands. There are overhanging streches called sticky-ends on each strand. They form hydrogen
bonds with their complementary cut counterparts. This stickiness of the ends facilitates the
action of the enzyme DNA ligase.
1. Fragments of DNA can be separated by a technique of gel electrophoresis.
2. Electrophoresis is a technique used for the separation of substances of different ionic properties. If such substances having different electric charges are taken in a solution and if an electric current is passed through this solution, the charged particles also move either towards anode or cathode, depending upon their charges, viz. positively charged molecules move towards cathode and negatively charged molecules towards anode through a medium. Since the DNA fragments are negatively charged molecules, they can be separated by allowing them to move towards anode.
3. Recently, the most commonly used matrix in gel electrophoresis is agarose which is polysaccharide extracted from sea weeds. DNA fragments move towards the anode according to their molecular size through the agarose gel. Thus the smaller fragments move farther away as compared to larger fragments.
4. The separated DNA fragments can be observed only after staining them with a solution of ethidium bromide. The bright orange coloured bands of DNA can be seen under UV light because pure DNA fragments cannot be seen under visible light.
5. The bands are cut out from the gel and extracted by using a convenient technique. This step is called elution. The eluted DNA fragments are then purified and used in constructing recombinant DNA by joining them with cloning vectors.
(c) Fragments are now added to the medium containing the vector DNA. The sticky ends facilitate
the action of the enzyme ligase and join the source DNA to the vector.