TOOLS AND TECHNIQUES OF GENETIC ENGINEERING
Tools
Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to produce a recombinant DNA (rONA). The 'biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA, passenger DNA and host cells.
1. Enzymes. Anumber of specific kinds of enzymes are employed in genetic engineering.
These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes.
(i) Lysing enzymes. These enzymes are used for opening the cells to get DNA for genetic experiment. Bacterial cell wall is commonly dissolved with the help of lysozyme.
(ii) Cleaving enzymes. These enzymes are used for DNA molecules. Cleaving enzymes are of three types; exonucleases, endonucleases and restriction endonucleases.
(a) Exonuclease cut off nucleotides from 5' or 3' ends of DNA molecule.
(b) Endonucleases break DNA duplex at any point except the end.
(c) Restriction endonucleases cleave DNA duplex at specific points in such a way that they come to possess short single stranded free ends. For example, a restriction endonuclease ECOR-I ( from Escherichia coli ) recognizes the base sequences GAATTC/CTTAAG in DNA duplex and cleaves it's between G and A.
`->` (Restriction enzymes are obtained frombacteria. They are useful to bacteria because the enzyme bring about fragmentation of viral DNA without affecting the bacterial genome , This is an adaption against bacteiophages. Restriction enzyme (Eco R-I) was discovered by Arber, Smith & Nathans ( 1978 Nobel prize). These enzymes exist in many bacteria besides cleavage some restriction endonuclease, also have capability of modification Modification in the form of methylation, by methylation the bacterial DNA modifles and therfore protects it's own chromosomal DNA from cleavage by these restriction enzymes.
`->` Restriction enzymes are used in recombinanat DNA technology , because they can be used in vitro to recognize
and cleave within specific DNA sequence typically consisting of 4 to 8 nucleotides. This specific 4 to 8 nucleotide sequence is called restriction site and is usually palindromaic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.
As a result the DNA fregments produced by cleavage with these enzymes have short single stranded overhang at each end these kinds of ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule back together again.
Therefore by cutting two different DNA samples with the same resfridion enzyme and mixing the fragments together a recombinant DNA molecule can be generated.
Exceptionally, some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in blunt end or flush end.
Nomenclature of enzyme- The first letter used for the enzyme is the first letter of the bacterium genus name in Italics) then comes the first two letter of it's species (In Italics), next is the strain of the organism, last is Roman numerical signifing the order in which the enzymes were isolated from that strain of bacteria.
Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to produce a recombinant DNA (rONA). The 'biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA, passenger DNA and host cells.
1. Enzymes. Anumber of specific kinds of enzymes are employed in genetic engineering.
These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes.
(i) Lysing enzymes. These enzymes are used for opening the cells to get DNA for genetic experiment. Bacterial cell wall is commonly dissolved with the help of lysozyme.
(ii) Cleaving enzymes. These enzymes are used for DNA molecules. Cleaving enzymes are of three types; exonucleases, endonucleases and restriction endonucleases.
(a) Exonuclease cut off nucleotides from 5' or 3' ends of DNA molecule.
(b) Endonucleases break DNA duplex at any point except the end.
(c) Restriction endonucleases cleave DNA duplex at specific points in such a way that they come to possess short single stranded free ends. For example, a restriction endonuclease ECOR-I ( from Escherichia coli ) recognizes the base sequences GAATTC/CTTAAG in DNA duplex and cleaves it's between G and A.
`->` (Restriction enzymes are obtained frombacteria. They are useful to bacteria because the enzyme bring about fragmentation of viral DNA without affecting the bacterial genome , This is an adaption against bacteiophages. Restriction enzyme (Eco R-I) was discovered by Arber, Smith & Nathans ( 1978 Nobel prize). These enzymes exist in many bacteria besides cleavage some restriction endonuclease, also have capability of modification Modification in the form of methylation, by methylation the bacterial DNA modifles and therfore protects it's own chromosomal DNA from cleavage by these restriction enzymes.
`->` Restriction enzymes are used in recombinanat DNA technology , because they can be used in vitro to recognize
and cleave within specific DNA sequence typically consisting of 4 to 8 nucleotides. This specific 4 to 8 nucleotide sequence is called restriction site and is usually palindromaic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.
As a result the DNA fregments produced by cleavage with these enzymes have short single stranded overhang at each end these kinds of ends are called sticky or cohesive ends because base pairing between them can stick the DNA molecule back together again.
Therefore by cutting two different DNA samples with the same resfridion enzyme and mixing the fragments together a recombinant DNA molecule can be generated.
Exceptionally, some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in blunt end or flush end.
Nomenclature of enzyme- The first letter used for the enzyme is the first letter of the bacterium genus name in Italics) then comes the first two letter of it's species (In Italics), next is the strain of the organism, last is Roman numerical signifing the order in which the enzymes were isolated from that strain of bacteria.
