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


● 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.


● `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' "`


● `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"`.


● 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.