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Chapter- 11, Biotechnology- Principles and Processes, (v) Restriction enzymes belong to a class of enzymes called, nucleases., Nucleases are of two types:, Exonucleases:-They remove nucleotides from the ends., Endonucleases:- They cut at specific positions within the, DNA., (a) Each restriction endonuclease recognises a specific, palindromic nucleotide sequences in the DNA., (b) Palindrome in DNA is a. sequence of base pairs that reads, same on the two strands when orientation of reading is kept, same., For example, the following sequences reads the same on the, two strands in 5′ -> 3′ direction as well as 3′ -> 5′ direction., 5′ — GAATTC — 3′, 3′ — CTTAAG — 5′, (vi) Mechanism of Action of Restriction Enzymes
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(a) Restriction enzymes cut the strand of DNA a little away from, the centre of the palindrome sites, but between the same two, bases on the opposite strands., (b) This leaves single stranded portions at the ends., (c) There are overhanging stretches called sticky ends on each, strands as given in above figure. These are named so, because, they form hydrogen bonds with their complementary cut, counterparts., (d) The stickiness of the ends facilitates the action of the, enzyme DNA ligase., (e) Restriction endonucleases are used in genetic engineering, to form recombinant molecules of DNA, which are composed of, DNA from different sources/genomes., (f) These sticky ends are complementary to each other when, cut by same restriction enzyme, therefore can be joined, together (end-to-end) using DNA ligases., Separation and Isolation of DNA Fragments, (i) The cutting of DNA by restriction’endonucleases results in, the fragments of DNA., (ii) The technique, which separates DNA fragments based on, their size is called gel electrophoresis., (iii) DNA fragments are negatively charged molecules. They, can be separated by forcing them to move towards the anode, under an electric field through a medium/matrix., (iv) The most common medium used is agarose, a natural, polymer extracted from sea weeds., (v) The DNA fragments separate (resolve) according to their, size through sieving effect provided by the agarose gel. The, smaller the fragment size, the farther it moves.
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(vi) The separated DNA fragments can be visualised only after, staining the DNA with a compound known as ethidium bromide, followed by exposure to UV radiation., (vii) The DNA fragments can be seen as bright orange coloured, bands. These separated bands are cut out from the agarose, gel and extracted from the gel piece. This is called elution., (viii) The purified DNA fragments can be used in constructing, recombinant DNA by joining them with cloning vectors., Cloning vectors, They are the DNA molecules that can carry a foreign DNA, segment into the host cell., (i) The vectors used in recombinant DNA technology can be:, (a) Plasmids Autonomously replicating circular, extra-chromosomal DNA., (b) Bacteriophages Viruses infecting bacteria., (c) Cosmids Hybrid vectors derived from plasmids which, contain cos site of X phage.
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(ii) Copy number can be defined as the number of copies of, vectors present in a cell., (iii) Bacteriophages have high number per cell, so their copy, number is also high in genome., (iv) Plasmids have only one or two copies per cell., (v) Copy number can vary from 1-100 or more than 100 copies, per cell., (vi) If an alien piece of DNA is linked with bacteriophage or, plasmid DNA, its number can be multiplied equal to the copy, number of the plasmid or bacteriophage., (vii) Features Required to Facilitate Cloning into Vector, (a) Origin of replication (Ori), (b) Selectable marker, (c) Cloning sites, (d) Vectors for cloning genes in plants and animals.
