*Berg is often considered "father of genetic engineering" He was awarded Nobel Prize in 1980.
definition of biotechnology which covers hoth traditional views and m biotechnology has been given hy European Federation of Biotechnology (E to EFB. "Biotechnology is the integrated use of biochemistry, microbiology and engine sciences in onder to achieve technologica! (industrial) application of th microorganisms, culured zissuescells and paris thereof" (EFB). According capabilities f Thus definition of biotechnology involves two common factors. First the use of biolo cal agents and second the product or service is generated for the well being of human.
Two Main Techniques of Modern Biotechnology-:The two main techniques thata birth to modern biotechnology are as follows:
(a) Genetic Engineering. It includes techniques to alter the nature of genetic material (DNA and RNA) to introduce these into host organisms and thus change the phonotype of the host organism.
(b) Chemical Engineering. It involves maintenancc of stcrilc microbial containination frec condition in chemical engineering processes to have growth of only the desired mi organism/eukaryotic cell in large quantities for the manufacture of biotechnological products such as antibioties, vaccines, enzymes, medicines, hormoncs, etc.
Role of Paul Berg. In 1972 genetic engineering was started by Paul Berg. Berę (1972) was abl Berg is uften considercd "father of genetic engincering". He was awarded Nobel 1980 e to introduce a gene of SV-40 virus into a bacterium with the help of lambda phage.
The technique of genetic engineering includes i) Formation of 'recembinant DNA' (rDNA). (ii) Use of gene cloning (iii) Gene transfer. It permits to isolate and introducc only one or a set of desirable genes without introducing undesirable genes into the target organism
A piece of DNA which is introduced into the alien (foreign) organism would not be ble of to multiply itself in the organism but when it gets incorporated into the genetic materia the recipient, it may multiply and be inherited alongwith the host DNA, because the alien pieceDNA has become part of chromosome which possesses the ability to replicate.There specific DNA sequence called the origin of replication in a chromosome th replicate and multiply itself in the host organism.It is also called the cloning,
Three basic step in creating genetically modified organisms (GMO)or transgenic organism.GMO contains a foreign gene/sigment of DNA.These three basic steps as follows.
(i) identification of DNA with desirable genes.
(ii) introduction of the identified DNA into the host.
(iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
(A) Enzyme.(B)Coloning Vectors (Vehicle DNA).(C) component host (for transformation with Recombinant DNA).
(A) Enzyme:
(1)Lysing Enzyme. this enzyme are used to open up the cell to get DNA for genetic experimentst. Lysozyme is usually used to dissolve the bacterial cell wall. In plant, cell wall is made up of cellulose, oil in fungi, it it is made aap of chitinase.
(2) cleaving enzyme. These enzymes are used to break DNA molecules. They are of three types-exonuclease, Indo nucleus and restriction endonuclease.
(a) exonuclease, they remove nucleotides from the terminal ends (either 5' or 3') of DNA in one strand of duplex.(b) Endonuclease, they make cards at specific position within the DNA. These enzymes do not cleave the ends and involve only on stand of the DNA duplex. (C) Restriction endonuclease, Descartes DNA duplex at specific points. Theiy are single stranded DNA are called sticky ends which can be joined end to end by DNA ligase.
RESTRICTION ENDONUCLEASE-The molecular scissor:
Restriction endonuclease was isolated for the first time by W. Arber in 1962 in bacteria.They are called "molecular scissors or biological scissors". In 1978 Arber, Smith and Nathan were awarded the Nobel Prize for the discovery of restriction endonuclease. They the base sequence at palindrome sites in DNA duplex and cut its strands. For iz estriction endonuclease EcoR 1 found in the colon bacteria E.coli, recognizes the base sequene GAATTC in DNA duplex and cuts its strands between G and A as shown bellow:
5'-GAATTC-3'
3'-CTTAAG-5'
only restriction enzymes type II are used in gene manipulation for two reasons. (a) No ATP is needed for the cleaving action. (b) It makes cleavage or cut in both the strands of DNA molecule.
Three main types of restriction endonu- cleases are type I, type II and type III Type I Restriction Endonucleases. These enzymes consist of 3 different subunits. They require ATP, Mg2+ and S-adenosyl methionine for restriction. Type I restriction endo- nucleases recognize specific sites within the DNA but do not cut these sites. Thus they are not used in recombinant DNA technology Type II Restriction Endonucleases. These enzymes are simple and require Mg2* ion or restriction. Out of the three types, only type II restriction enzymes are used in recombinant DNA technology. ype III Restriction Endonucleases. These enzymes consist of two different subunits. The y require ATP, Mg2+ and S-adenosyl methionine for restriction. They recognize specific es within DNA but do not cut these sites, therefore, these restriction endonucleases are hot used and type II. in recombinant DNA technology. They have intermediate properties between type I & type II.
DEFINITION OF BIOTECHNOLOGY:
definition of biotechnology which covers hoth traditional views and m biotechnology has been given hy European Federation of Biotechnology (E to EFB. "Biotechnology is the integrated use of biochemistry, microbiology and engine sciences in onder to achieve technologica! (industrial) application of th microorganisms, culured zissuescells and paris thereof" (EFB). According capabilities f Thus definition of biotechnology involves two common factors. First the use of biolo cal agents and second the product or service is generated for the well being of human.
I. PRINCIPLES OF BIOTECHNOLOGY :
Two Main Techniques of Modern Biotechnology-:The two main techniques thata birth to modern biotechnology are as follows:
(a) Genetic Engineering. It includes techniques to alter the nature of genetic material (DNA and RNA) to introduce these into host organisms and thus change the phonotype of the host organism.
(b) Chemical Engineering. It involves maintenancc of stcrilc microbial containination frec condition in chemical engineering processes to have growth of only the desired mi organism/eukaryotic cell in large quantities for the manufacture of biotechnological products such as antibioties, vaccines, enzymes, medicines, hormoncs, etc.
1. Conceptual Development of the Principles of Genetic Engineering:
Genetic engineering is a kind of biotechnology which deals with the manipulation genetic material by man in vitro Twn Main Discoveries. Genetic engineering is based on two important discoveries in bacteria.
(i) Presence of plasmids in bacteria which can undergo replication alongwith and independent of chromosomal DNA (ii) Restriction endonucleases (Arber, Nathan and Smith 1970; Nobel Prize in 1978) which can break DNA at specific sites. They are appropriately called molecular scissors or biological scissors.Role of Paul Berg. In 1972 genetic engineering was started by Paul Berg. Berę (1972) was abl Berg is uften considercd "father of genetic engincering". He was awarded Nobel 1980 e to introduce a gene of SV-40 virus into a bacterium with the help of lambda phage.
The technique of genetic engineering includes i) Formation of 'recembinant DNA' (rDNA). (ii) Use of gene cloning (iii) Gene transfer. It permits to isolate and introducc only one or a set of desirable genes without introducing undesirable genes into the target organism
A piece of DNA which is introduced into the alien (foreign) organism would not be ble of to multiply itself in the organism but when it gets incorporated into the genetic materia the recipient, it may multiply and be inherited alongwith the host DNA, because the alien pieceDNA has become part of chromosome which possesses the ability to replicate.There specific DNA sequence called the origin of replication in a chromosome th replicate and multiply itself in the host organism.It is also called the cloning,
2. Construction of the First Artificial Recombinant DNA Molecule:
.at is respon- e for initiating replication.Thus, an alien DNA linked with the origin of replication, can .e., forming multiple identical copies of any template DNA The first recombinant DNA was constructed by Stanley Cohen and Herbert Boyer in hey cut the piece of DNA from a plasmid carrying antibiotic resistancegene in the bacterium Salmonella typhimurium.Cutting of a piece of DNA from a plasmid was with the help of restriction enzymes (also called molecular scissors or chemical scalpels).The piece of foreign DNA cut from the plasmid was linked with the plasmid DNA acting as vector.Linking of the piece of foreign DNA with vcctor was done with the help of the ly formed DNA having integrated fragment of antibiotic resistant gene is called recombinant DNA.The vector is used to transfer, recombinant DNA to E.coli.This transfer of recom- binant DNA is similar to the transfer of malarial parasite from diseased person into the healthy person through female Anopheles mosquito (acts as an insect vector).When this recombi nant DNA is transferred into Escherichia coli, it could replicate in the new host cell in the presence of DNA polymerase enzyme and make multiple copies of recombinant DNA.The ability to multiply copies of antibiotic resistance gene in E. coli was termed as cloning o enzyme DNA ligase which acts on cut DNA molecules and join their ends.This new antibiotic resistance gene in E. coli.Three basic step in creating genetically modified organisms (GMO)or transgenic organism.GMO contains a foreign gene/sigment of DNA.These three basic steps as follows.
(i) identification of DNA with desirable genes.
(ii) introduction of the identified DNA into the host.
(iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
2.TOOLS OF RECOMBINANT DNA TECHNOLOGY:
Three types of "biological tools" are used in the formation of Recombinant DNA.These are follows:(A) Enzyme.(B)Coloning Vectors (Vehicle DNA).(C) component host (for transformation with Recombinant DNA).
(A) Enzyme:
(1)Lysing Enzyme. this enzyme are used to open up the cell to get DNA for genetic experimentst. Lysozyme is usually used to dissolve the bacterial cell wall. In plant, cell wall is made up of cellulose, oil in fungi, it it is made aap of chitinase.
(2) cleaving enzyme. These enzymes are used to break DNA molecules. They are of three types-exonuclease, Indo nucleus and restriction endonuclease.
(a) exonuclease, they remove nucleotides from the terminal ends (either 5' or 3') of DNA in one strand of duplex.(b) Endonuclease, they make cards at specific position within the DNA. These enzymes do not cleave the ends and involve only on stand of the DNA duplex. (C) Restriction endonuclease, Descartes DNA duplex at specific points. Theiy are single stranded DNA are called sticky ends which can be joined end to end by DNA ligase.
RESTRICTION ENDONUCLEASE-The molecular scissor:
Restriction endonuclease was isolated for the first time by W. Arber in 1962 in bacteria.They are called "molecular scissors or biological scissors". In 1978 Arber, Smith and Nathan were awarded the Nobel Prize for the discovery of restriction endonuclease. They the base sequence at palindrome sites in DNA duplex and cut its strands. For iz estriction endonuclease EcoR 1 found in the colon bacteria E.coli, recognizes the base sequene GAATTC in DNA duplex and cuts its strands between G and A as shown bellow:
5'-GAATTC-3'
3'-CTTAAG-5'
only restriction enzymes type II are used in gene manipulation for two reasons. (a) No ATP is needed for the cleaving action. (b) It makes cleavage or cut in both the strands of DNA molecule.
1) Types of Restriction Endonucleases.
Three main types of restriction endonu- cleases are type I, type II and type III Type I Restriction Endonucleases. These enzymes consist of 3 different subunits. They require ATP, Mg2+ and S-adenosyl methionine for restriction. Type I restriction endo- nucleases recognize specific sites within the DNA but do not cut these sites. Thus they are not used in recombinant DNA technology Type II Restriction Endonucleases. These enzymes are simple and require Mg2* ion or restriction. Out of the three types, only type II restriction enzymes are used in recombinant DNA technology. ype III Restriction Endonucleases. These enzymes consist of two different subunits. The y require ATP, Mg2+ and S-adenosyl methionine for restriction. They recognize specific es within DNA but do not cut these sites, therefore, these restriction endonucleases are hot used and type II. in recombinant DNA technology. They have intermediate properties between type I & type II.
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