GENETIC ENGINEERING
UNIT 1
INTRODUCTION:
Genetic engineering stands for the transfer of DNA between hosts (or species) by in
vitro enzymatic manipulation.
This means that the DNA to be transferred, will be duplicated in the new host. Since
most DNA fragments are incapable of self-replication in E.coli or any other host cell,
an additional segments of DNA, capable of autonomous replication must be linked to
the fragments to be cloned.
This autonomously replication fragments is the molecular cloning vector. Thus
vectors are self-replicating DNA molecules into which foreign DNA (gene of interest)
is inserted.
They play an important role in recombinant DNA technology.
Most cloning vectors are originally derived from naturally occurring
extrachromosomal elements such as bacteriophage and plasmids.
Stanley cohen and coworkers (1975) first reported the use of bacterial plasmids as
molecular cloning vectors.
Currently, specialized cloning vectors are available that enable the investigator to:
1. Identify and isolate regulatory DNA sequences such as promoters and
terminators
2. Identify open translation reading frames
3. Overproduce useful RNAs and proteins and
4. Determine the nucleotide sequence of genes and segments of DNA.
VECTORS:
• Def: the DNA that carries the desired gene to the host cell is called gene cloning
vector/cloning vector/vector/cloning vehicle or carrier DNA.
• Plasmid consist of a replicon (i.e., unit of genetic material capable of
independent replication/ self-replication) that is stable inherited (maintained
without specific selection) in an extra chromosomal state.
• Plasmids, viral DNAs and cosmids are used as gene cloning vectors.
• Plasmids: are circular, dsDNA usually present in prokaryotic cells. They can
carry a foreign DNA of 5-15 kbp size. Eg: pBR322, Ti plasmids (carry genes to
plant cells).
1
, GENETIC ENGINEERING
• Viral DNAs: linear/circular and ds/ss. It can carry DNAs of 10-25 kbp size. Eg: l
phage and M13 phage ( carry genes to bacteria), simian virus 40 (SV 40-carry
genes to animal cells).
• Cosmids: type of constructed plasmids containing ss sites (cos-sites) of l DNA.
They can carry DNAs of 25-45kbp size to bacteria. Eg : pHV79.
Why vectors are used?
The desired gene may be hydrolyzed by cellular enzymes immediately after the
introduction. Further, the chances for its expression is also very poor. In order to
overcome these problems, the desired gene is inserted into suitable vector for gene
cloning.
CHARACTERISTICS OF AN IDEAL VECTORS:
1. It must be small in size.
2. It must be self-replicating inside host cell.
3. It must possess restriction site for Restriction Endonuclease enzymes and ori
region.
4. Introduction of donor DNA fragment must not interfere with replication
property of the vector.
5. It must possess some marker gene such that it can be used for later
identification of recombinant cell.
6. It must possess multiple cloning site or polylinker.
7. It should contain specific control systems like promoters, terminators, ribosome
binding sites, etc . so that the cloned DNA should express properly.
NOTE:
During the early 1970s, Stanley cohen and herb boyer selected E.coli starin K12 for
conducting research work on molecular biology.
Since then E.coli K12 has been used for various experiments in recombinant DNA
technology and production of products. One of the unique features of E.coli is that it
is found in human intestine and cannot thrive outside the laboratory environment.
E.coli strain K12 mutant in restriction-modification system is used in gene cloning.
E.coli strain K12 mutant in endH gene (encodes DNA specific endonucleases )
increases the yield of plasmid DNA and improves DNA quality.
2
, GENETIC ENGINEERING
The recA gene is associated with expression of recombination proteins. Therefore,
E.coli strain K12 mutant in recA gene ensures about the biosafety of E.coli strain K12
because they can easily be killed by UV light.
PLASMIDS:
• Plasmids are small, circular, ds, extrachromosomal DNAs present in the
bacterial cells (not found free)
• Plasmids are independent of chromosomal DNA.
• It depends on host proteins for replication, maintenance, and other functions.
• They are present in a characteristic number of copies in a bacterial cell, yeast
cell, in eukaryotic organelles such as mitochondria.
• They replicate independently due to presence of ori region.
• They are 1 kbp-200 kbp in size and have limited number of genes.
• Copy no: the no. of plasmids present in the bacterial cell .
• Amplification of plasmid: The copy number of a plasmid can be increased
manifold experimentally. This process is called amplification of the plasmid
DNA. This property has been exploited to increase the plasmid copy number.
• The copy number of plasmids usually varies from 1 to 50, it can be further
increased upto 3000 by treating the bacterial culture with chloramphenicol at
exponential phase of the bacterial culture.( Adding chloramphenicol arrests
chromosomal DNA replication and cell division, but the plasmid will continue
replicating. This will result in many more copies of your vector per bacterial
genome). This helps in easy isolation of plasmids.
• The term plasmid was first introduced by the American molecular
biologist Joshua Lederberg in 1952.
3
UNIT 1
INTRODUCTION:
Genetic engineering stands for the transfer of DNA between hosts (or species) by in
vitro enzymatic manipulation.
This means that the DNA to be transferred, will be duplicated in the new host. Since
most DNA fragments are incapable of self-replication in E.coli or any other host cell,
an additional segments of DNA, capable of autonomous replication must be linked to
the fragments to be cloned.
This autonomously replication fragments is the molecular cloning vector. Thus
vectors are self-replicating DNA molecules into which foreign DNA (gene of interest)
is inserted.
They play an important role in recombinant DNA technology.
Most cloning vectors are originally derived from naturally occurring
extrachromosomal elements such as bacteriophage and plasmids.
Stanley cohen and coworkers (1975) first reported the use of bacterial plasmids as
molecular cloning vectors.
Currently, specialized cloning vectors are available that enable the investigator to:
1. Identify and isolate regulatory DNA sequences such as promoters and
terminators
2. Identify open translation reading frames
3. Overproduce useful RNAs and proteins and
4. Determine the nucleotide sequence of genes and segments of DNA.
VECTORS:
• Def: the DNA that carries the desired gene to the host cell is called gene cloning
vector/cloning vector/vector/cloning vehicle or carrier DNA.
• Plasmid consist of a replicon (i.e., unit of genetic material capable of
independent replication/ self-replication) that is stable inherited (maintained
without specific selection) in an extra chromosomal state.
• Plasmids, viral DNAs and cosmids are used as gene cloning vectors.
• Plasmids: are circular, dsDNA usually present in prokaryotic cells. They can
carry a foreign DNA of 5-15 kbp size. Eg: pBR322, Ti plasmids (carry genes to
plant cells).
1
, GENETIC ENGINEERING
• Viral DNAs: linear/circular and ds/ss. It can carry DNAs of 10-25 kbp size. Eg: l
phage and M13 phage ( carry genes to bacteria), simian virus 40 (SV 40-carry
genes to animal cells).
• Cosmids: type of constructed plasmids containing ss sites (cos-sites) of l DNA.
They can carry DNAs of 25-45kbp size to bacteria. Eg : pHV79.
Why vectors are used?
The desired gene may be hydrolyzed by cellular enzymes immediately after the
introduction. Further, the chances for its expression is also very poor. In order to
overcome these problems, the desired gene is inserted into suitable vector for gene
cloning.
CHARACTERISTICS OF AN IDEAL VECTORS:
1. It must be small in size.
2. It must be self-replicating inside host cell.
3. It must possess restriction site for Restriction Endonuclease enzymes and ori
region.
4. Introduction of donor DNA fragment must not interfere with replication
property of the vector.
5. It must possess some marker gene such that it can be used for later
identification of recombinant cell.
6. It must possess multiple cloning site or polylinker.
7. It should contain specific control systems like promoters, terminators, ribosome
binding sites, etc . so that the cloned DNA should express properly.
NOTE:
During the early 1970s, Stanley cohen and herb boyer selected E.coli starin K12 for
conducting research work on molecular biology.
Since then E.coli K12 has been used for various experiments in recombinant DNA
technology and production of products. One of the unique features of E.coli is that it
is found in human intestine and cannot thrive outside the laboratory environment.
E.coli strain K12 mutant in restriction-modification system is used in gene cloning.
E.coli strain K12 mutant in endH gene (encodes DNA specific endonucleases )
increases the yield of plasmid DNA and improves DNA quality.
2
, GENETIC ENGINEERING
The recA gene is associated with expression of recombination proteins. Therefore,
E.coli strain K12 mutant in recA gene ensures about the biosafety of E.coli strain K12
because they can easily be killed by UV light.
PLASMIDS:
• Plasmids are small, circular, ds, extrachromosomal DNAs present in the
bacterial cells (not found free)
• Plasmids are independent of chromosomal DNA.
• It depends on host proteins for replication, maintenance, and other functions.
• They are present in a characteristic number of copies in a bacterial cell, yeast
cell, in eukaryotic organelles such as mitochondria.
• They replicate independently due to presence of ori region.
• They are 1 kbp-200 kbp in size and have limited number of genes.
• Copy no: the no. of plasmids present in the bacterial cell .
• Amplification of plasmid: The copy number of a plasmid can be increased
manifold experimentally. This process is called amplification of the plasmid
DNA. This property has been exploited to increase the plasmid copy number.
• The copy number of plasmids usually varies from 1 to 50, it can be further
increased upto 3000 by treating the bacterial culture with chloramphenicol at
exponential phase of the bacterial culture.( Adding chloramphenicol arrests
chromosomal DNA replication and cell division, but the plasmid will continue
replicating. This will result in many more copies of your vector per bacterial
genome). This helps in easy isolation of plasmids.
• The term plasmid was first introduced by the American molecular
biologist Joshua Lederberg in 1952.
3
