Genetic Engineering
➔ Genetic engineering is the directed manipulation of the hereditary material.
➔ There are several other terms that can be used to describe the technology, including
gene manipulation, gene cloning, recombinant DNA technology, genetic modification,
and the new genetics.
➔ It is based on a set of molecular techniques collectively called recombinant DNA (rDNA)
technology. In essence, rDNA technology consists of the splicing of a piece of DNA to a
suitable carrier which is then introduced in a convenient cell that allows the ‘passenger’
or ‘foreign’ DNA to express itself in the new surroundings.
➔ This field has grown rapidly to the point where, in many laboratories around the world, it
is now routine practice to isolate a specific DNA fragment from the genome of an
organism, determine its base sequence, and assess its function.
➔ The technology is also now used in many other applications, including forensic analysis
of scene-of-crime samples, paternity disputes, medical diagnosis, genome mapping and
sequencing, and the biotechnology industry.
,RECOMBINANT DNA TECHNOLOGY
➔ The birth of rDNA technology is based on two sets of discoveries: the discovery of the
Watson and Crick DNA model together with the studies that ensued, and the
identification of enzymes that can cleave, elongate, join or otherwise modify DNA
molecules.
➔ These two discoveries complemented each other, and opened up avenues of biological
research in an unprecedented manner. It became possible to snip out a piece of DNA
from any organism and prepare it for amplification in a conveniently handled cell.
➔ A piece of DNA is unable to maintain or replicate itself on its own unless it possesses
certain essential features.
➔ To clone such a DNA fragment it has to be first joined to another DNA that possesses
these essential features. The first job in gene cloning is to find a suitable carrier or vector
DNA for the passenger DNA.
➔ The DNA containing the fragment of choice is first cleaved into several pieces.
➔ This collection of fragments is then mixed with the vector DNA which also has two free
ends.
➔ Both types of DNA fragments join with each other forming several kinds of hybrid
molecules each containing a particular fragment from the donor DNA.
➔ The linear hybrid molecules are then circularized and the cut ends sealed.
➔ The next step is to look for the hybrids that contain the passenger DNA.
➔ To do this, the total population of hybrid and non-hybrid DNA pieces are first introduced
into suitable cells that grow and replicate without undue fuss.
➔ The cell of choice to date has been that of the bacterium E. coli. A few out of thousands
of DNA pieces manage to enter these cells.
➔ The latter are said to be ‘transformed’ (due to the addition of extrinsic genetic matter).
Each transformed cell grows a colony of its own, in which every member is genetically
alike.
➔ Out of the many colonies that surface, only some contain the DNA fragment of interest.
➔ These colonies have to be distinguished and recultured separately.
➔ After selecting the clones carrying the desired hybrid molecules they are cultured
extensively to provide sufficient numbers of cells from which a worthwhile quantity of the
hybrid DNA can be extracted.
➔ The recombinant DNA is then extracted from lysed cells, purified and used as desired.
➔ The first gene was cloned in 1973 by Herbert Boyer and Stanley Cohen of Stanford
University, California.
➔ The year before, the first hybrid DNA was produced by Janet Merty and Ron David also
at Stanford.
➔ Both these successes rested on the discoveries of two unique enzymes: ligase in 1967
by Merty and David and restriction enzymes in 1970 by Hamilton O. Smith.
➔ Ligase joins DNA backbones; restriction enzymes cleave the DNA at regions that are
specific for each enzyme.
➔ The specificity lies in a set of 4–7 base-pairs that are recognized by a particular enzyme
which then cleaves the DNA backbone within or very near this recognition sequence.
➔ The utility of this technique has been very much enhanced by the discovery of other
enzymes that catalyze other reactions such as DNA and RNA polymerases,
exonucleases and RNA and DNA nucleases that act on single- or double-stranded
molecules.
➔ One of the enzymes that has proved to be invaluable in rDNA technology is reverse
transcriptase, which can mediate the synthesis of a DNA strand along an RNA template.
, It is used as a tool for making copies from cell RNA molecules of genes that are not
easily located or reached.
➔ Three experimental techniques that have contributed to advances in DNA biology are gel
electrophoresis, Southern blotting and DNA sequencing.
➔ A fourth one that has become possible due to these techniques and appears to be a
promising tool is site-directed mutagenesis.
➔ The latest invaluable technique for DNA engineering is the one known as the
Polymerase Chain Reaction or the PCR technique.
➔ DNA fragments and proteins of various sizes can be fractionated elegantly by gel
electrophoresis.
➔ The ability to separate fragments of DNA differing by only one nucleotide is utilized
profitably in methods for DNA sequencing, which lays bare the nucleotide pattern in a
DNA molecule.
HOW TO CLONE A GENE
➔ The operations involved in gene cloning consist of the following steps:
1. Cut the larger DNA (donor) and the vector DNA with the same special enzyme. Join a
donor and a vector DNA. A recombinant or rDNA has been made. One of these rDNAs
has the fragment with a desired gene G.
2. Amplify (clone) each rDNA in E. coli cells.
3. Select clones with rDNA. Some of them carry G.
4. Select clones with G.
5. Increase the number of selected clones. All cells have rDNA with G.
6. Extract rDNA from cells, every rDNA has G.
7. Use the rDNA for research or application.
➔ Genetic engineering is the directed manipulation of the hereditary material.
➔ There are several other terms that can be used to describe the technology, including
gene manipulation, gene cloning, recombinant DNA technology, genetic modification,
and the new genetics.
➔ It is based on a set of molecular techniques collectively called recombinant DNA (rDNA)
technology. In essence, rDNA technology consists of the splicing of a piece of DNA to a
suitable carrier which is then introduced in a convenient cell that allows the ‘passenger’
or ‘foreign’ DNA to express itself in the new surroundings.
➔ This field has grown rapidly to the point where, in many laboratories around the world, it
is now routine practice to isolate a specific DNA fragment from the genome of an
organism, determine its base sequence, and assess its function.
➔ The technology is also now used in many other applications, including forensic analysis
of scene-of-crime samples, paternity disputes, medical diagnosis, genome mapping and
sequencing, and the biotechnology industry.
,RECOMBINANT DNA TECHNOLOGY
➔ The birth of rDNA technology is based on two sets of discoveries: the discovery of the
Watson and Crick DNA model together with the studies that ensued, and the
identification of enzymes that can cleave, elongate, join or otherwise modify DNA
molecules.
➔ These two discoveries complemented each other, and opened up avenues of biological
research in an unprecedented manner. It became possible to snip out a piece of DNA
from any organism and prepare it for amplification in a conveniently handled cell.
➔ A piece of DNA is unable to maintain or replicate itself on its own unless it possesses
certain essential features.
➔ To clone such a DNA fragment it has to be first joined to another DNA that possesses
these essential features. The first job in gene cloning is to find a suitable carrier or vector
DNA for the passenger DNA.
➔ The DNA containing the fragment of choice is first cleaved into several pieces.
➔ This collection of fragments is then mixed with the vector DNA which also has two free
ends.
➔ Both types of DNA fragments join with each other forming several kinds of hybrid
molecules each containing a particular fragment from the donor DNA.
➔ The linear hybrid molecules are then circularized and the cut ends sealed.
➔ The next step is to look for the hybrids that contain the passenger DNA.
➔ To do this, the total population of hybrid and non-hybrid DNA pieces are first introduced
into suitable cells that grow and replicate without undue fuss.
➔ The cell of choice to date has been that of the bacterium E. coli. A few out of thousands
of DNA pieces manage to enter these cells.
➔ The latter are said to be ‘transformed’ (due to the addition of extrinsic genetic matter).
Each transformed cell grows a colony of its own, in which every member is genetically
alike.
➔ Out of the many colonies that surface, only some contain the DNA fragment of interest.
➔ These colonies have to be distinguished and recultured separately.
➔ After selecting the clones carrying the desired hybrid molecules they are cultured
extensively to provide sufficient numbers of cells from which a worthwhile quantity of the
hybrid DNA can be extracted.
➔ The recombinant DNA is then extracted from lysed cells, purified and used as desired.
➔ The first gene was cloned in 1973 by Herbert Boyer and Stanley Cohen of Stanford
University, California.
➔ The year before, the first hybrid DNA was produced by Janet Merty and Ron David also
at Stanford.
➔ Both these successes rested on the discoveries of two unique enzymes: ligase in 1967
by Merty and David and restriction enzymes in 1970 by Hamilton O. Smith.
➔ Ligase joins DNA backbones; restriction enzymes cleave the DNA at regions that are
specific for each enzyme.
➔ The specificity lies in a set of 4–7 base-pairs that are recognized by a particular enzyme
which then cleaves the DNA backbone within or very near this recognition sequence.
➔ The utility of this technique has been very much enhanced by the discovery of other
enzymes that catalyze other reactions such as DNA and RNA polymerases,
exonucleases and RNA and DNA nucleases that act on single- or double-stranded
molecules.
➔ One of the enzymes that has proved to be invaluable in rDNA technology is reverse
transcriptase, which can mediate the synthesis of a DNA strand along an RNA template.
, It is used as a tool for making copies from cell RNA molecules of genes that are not
easily located or reached.
➔ Three experimental techniques that have contributed to advances in DNA biology are gel
electrophoresis, Southern blotting and DNA sequencing.
➔ A fourth one that has become possible due to these techniques and appears to be a
promising tool is site-directed mutagenesis.
➔ The latest invaluable technique for DNA engineering is the one known as the
Polymerase Chain Reaction or the PCR technique.
➔ DNA fragments and proteins of various sizes can be fractionated elegantly by gel
electrophoresis.
➔ The ability to separate fragments of DNA differing by only one nucleotide is utilized
profitably in methods for DNA sequencing, which lays bare the nucleotide pattern in a
DNA molecule.
HOW TO CLONE A GENE
➔ The operations involved in gene cloning consist of the following steps:
1. Cut the larger DNA (donor) and the vector DNA with the same special enzyme. Join a
donor and a vector DNA. A recombinant or rDNA has been made. One of these rDNAs
has the fragment with a desired gene G.
2. Amplify (clone) each rDNA in E. coli cells.
3. Select clones with rDNA. Some of them carry G.
4. Select clones with G.
5. Increase the number of selected clones. All cells have rDNA with G.
6. Extract rDNA from cells, every rDNA has G.
7. Use the rDNA for research or application.
