Biology TOOLS OF RECOMBINANT DNA TECHNOLOGY

### KEY TOPICS

star Restriction Enzymes
star Nucleases
star Cutting the Restriction Sequences
star Separation and isolation of DNA fragments

### RESTRICTION ENZYMES

● In the year color{Violet}"1963", the color{Violet}"two enzymes" responsible for restricting the growth of color{Violet}"bacteriophage" in Escherichia coli were isolated.

● One of these color{Violet}"added methyl groups" to DNA, while the other color{Violet}"cut DNA".

● The later was called color{Brown}"restriction endonuclease".

● The color{Brown}"first restriction endonuclease"–color{Violet}"Hind II", whose functioning depended on a specific DNA nucleotide sequence was isolated and characterised color{Violet}"five years later".

● It was found that color{Violet}"Hind II" always cut DNA molecules at a particular point by recognising a color{Violet}"specific sequence" of six base pairs.

● This color{Violet}"specific base sequence" is known as the color{Brown}"recognition sequence" for Hind II.

● Besides Hind II, today we know more than color{Violet}"900 restriction enzymes" that have been isolated from over color{Violet}"230 strains of bacteria" each of which recognise different recognition sequences

.
● The color{Violet}"convention for naming" these enzymes is the color{Violet}"first letter" of the name comes from the genes and the color{Violet}"second two letters" come from the species of the prokaryotic cell from which they were isolated.

● color{Brown}"E.g"., color{Violet}"EcoRI" comes from color{Violet}"Escherichia coli" color{Violet}"RY 13". In EcoRI, the letter ‘R’ is derived from the name of strain.

● color{Violet}"Roman numbers" following the names indicate the color{Violet}"order" in which the color{Violet}"enzymes were isolated" from that strain of bacteria.

### NUCLEASES

● color{Violet}"Restriction enzymes" belong to a larger class of enzymes called color{Brown}"nucleases".

● These are of color{Violet}"two kinds"; color{Brown}"exonucleases and endonucleases".

● color{Violet}"Exonucleases" remove nucleotides from the color{Violet}"ends of the DNA" whereas, color{Violet}"endonucleases" make cuts at specific positions color{Violet}"within the DNA".

● Each restriction endonuclease functions by color{Violet}"inspecting’ the length" of a DNA sequence.

● Once it finds its color{Violet}"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 backbone.

● Each restriction endonuclease recognises a color{Violet}"specific palindromic" color{Violet}"nucleotide sequences" in the DNA.

● These are groups of letters that form the color{Violet}"same words" when read both color{Violet}"forward and backward", e.g., color{Brown}"MALAYALAM".

● As against a word-palindrome where the same word is read in both directions, the color{Brown}"palindrome in DNA" is a sequence of base pairs that reads same on the color{Violet}"two strands" when orientation of reading is kept the same.

● Example, the following sequences color{Violet}"reads the same" on the two strands in color{Violet}"5'- 3' direction".

● This is also true if read in the color{Violet}"3' -5'" direction.

color{Blue}"5' —— GAATTC —— 3' "
color{Blue}"3' —— CTTAAG —— 5' "

### CUTTING THE RESTRICTION SEQUENCES

● color{Violet}"Restriction enzymes" cut the strand of DNA a color{Violet}"little away" from the centre of the palindrome sites, but color{Violet}"between the same" two bases on the opposite strands.

● This leaves color{Violet}"single stranded portions" at the ends.

● There are color{Violet}"overhanging stretches" called color{Brown}"sticky ends" on each strand.

● These are named so because they form color{Violet}"hydrogen bonds" with their color{Violet}"complementary cut counterparts".

● This color{Violet}"stickiness" of the ends facilitates the action of the color{Violet}"enzyme DNA ligase".

● Restriction endonucleases are used in color{Violet}"genetic engineering" to form color{Violet}"recombinant molecules" of DNA, which are composed of DNA from different sources/genomes.

● When cut by the color{Violet}"same restriction enzyme", the resultant DNA fragments have the color{Violet}"same kind of" color{Violet}"sticky-ends" and, these can be joined together (end-to-end) using DNA ligases.

● It has to be realised that normally, unless one cuts the color{Violet}"vector and the source DNA" with the color{Violet}"same restriction enzyme", the recombinant vector molecule color{Violet}"cannot be created".

### SEPARATION AND ISOLATION OF DNA FRAGMENTS

● The cutting of DNA by color{Violet}"restriction endonucleases" results in the fragmentes of DNA.

● These fragments can be separated by a technique known as color{Brown}"gel electrophoresis".

● Since DNA fragments are color{Violet}"negatively charged molecules" they can be separated by forcing them to move towards the anode under an color{Violet}"electric field" through a medium/matrix.

● Nowadays the most commonly used color{Violet}"matrix is agarose" which is a natural polymer extracted from color{Violet}"sea weeds".

● The DNA fragments color{Violet}"separate (resolve)" according to their color{Violet}"size through" color{Violet}"sieving effect" provided by the agarose gel.

● Hence, the color{Violet}"smaller" the fragment size, the color{Violet}"farther" it moves.

● The separated DNA fragments can be color{Violet}"visualised" only after staining the DNA with a compound known as color{Violet}"ethidium bromide" followed by exposure to color{Violet}"UV radiation" (One cannot see pure DNA fragments in the visible light and without staining).

● One can see color{Violet}"bright orange" color{Violet}"coloured bands" of DNA in a ethidium bromide stained gel exposed to UV light.

● The separated bands of DNA are color{Violet}"cut out" from the agarose gel and color{Violet}"extracted" from the gel piece.

● This step is known as color{Violet}"elution".

● The DNA fragments purified in this way are used in color{Violet}"constructing recombinant DNA" by joining them with cloning vectors.