Cloning vectors
- the DNA molecule that can carry a foreign DNA segment and replicate inside. The host cell is called a vector the host cell.
(1) the vectors used in Recombinant DNA technology can be
(a) plasmids autonomously replicating circular extrachromosomal DNA.
(b) bacteriophages virus infecting bacteria.
(c) cosmids hybrid vectors drive from plasma cell which contain cos site of Lambda phage
(2) copy number can be defined as the number of copies of vectors present in a Cell So, in rDNA technology is used in vectors are selected which have high copy number it can vary from 1- 100 or more than 100 copies per cell .
bacteriophages have hi number per cells with their copy number is also High in genome.
plasmids have Only one or two copies per cell.
(3) if an Alien piece of DNA is linked with bacteriophage of plasmid. DNA its number can be multiplied equal to the copy number of the plasmids or bacteriophage.
(4) present day vectors help in easy linking of foreign DNA and selection of recombinants from non Recombinant.
FEATURES Required To facilitate cloning into vector :-
(a) origin of replication (ori) is a sequence from where replication starts.
* Any piece of DNA when linked to this sequence can be made to replicate within the host cells.
* The sequence is also responsible for controlling the copy number of the linked DNA.
* So if one wants to recover many copies of target DNA it should be cloned in a vector whose Origins support High copy number.
(b) selectable marker helps in identifying and eliminating non transformants and selectivity permitting the growth of the Transformants.
* Transformation is a process through which the piece of the DNA is introduced in the host bacterium. Those host cells which have got Recombinant DNA are known as transformants,while those have not got the Recombinant DNA known as non-transformants.
* The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc are some useful . Selectable markers for E.coli.
* The normal E.coli cells do not carry resistance against any of these antibiotics.
(c) cloning sites are required to link the alien DNA with the vector. It contains restriction sites.
* The vector requires very few or single recognition sites for the commonly used restriction enzymes.
* The presence of more than one recognition sites within the vector will generate several fragments leads to complication in gene cloning. So one recognition site for an enzyme is preferred.
* Ligation of alien DNA is carried out at a restriction site presents in one of the two antibiotic resistance genes, for example, ligating a foreign DNA at the Bam HI site of tetracycline resistance clean in the vector pBR322.
* The Recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but it still can be selected out from non Recombinant Once by plating in the Transformants on ampicillin containing medium.
* The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.
* The Recombinant will grow in ampicillin containing medium but not on the containing tetracycline because in Recombinant tetracycline antibiotic resistance gene is the disturbed.
* The non Recombinant will grow on the medium containing both the antibiotic because both antibiotic resistance genes are functional.
* In this example one antibiotic resistance gene helps in selecting the transformants whereas the other antibiotic resistance genes get inactivated due to insertion of alien DNA and helps in selection of recombinants.
* Selection of recombinants due to inactivation of antibiotic is cumbersome procedure, because it requires simultaneous plating on two plates having different antibiotics. So alternative selectable markers are developed available which differentiate recombinants from non recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
* In this method, a recombinant DNA is inserted within the coding sequence of an enzyme Beta-galactosidase. So, if genes of interest gets incorporated,
Beta - galactosidase gene will be disturbed.
* This results in inactivation of the enzyme Beta-galactosidase (insertional inactivation).
* The bacterial colonies whose plasmids do not have an insert to produce blue colour when grown on a chromogenic substrate because of the presence of Beta galactosidase enzyme the presence of an insert in bacterial colonies does not produce any colour due to insertional inactivation of gene producing beta-galactosidase enzyme.
(d) Vectors for cloning genes in plants and animals several vectors are used to clone genes of interest in plants and animals.
* In plants, The Tumor inducing plasmids of agrobacterium tumefaciens is used as a cloning vector. It is known as Natural engineer of plants.
* Agrobacterium tumefaciens is a pathogen of several dicot plants.
* The tumor inducing plasmids of this bacteria has now been modified into a cloning vector which is no more pathogenic to the plants. It delivers a piece of DNA known as T-DNA in the Ti-plasmid which transforms normal plant cells into tumors cells to produce Chemicals required by the pathogens.
* Retrovirus, adenovirus, papillomavirus are also used as cloning vectors in animals because of their ability to transform normal size into cancerous cells.
Competent host organisms :-
- It is required because DNA being a hydrophilic molecule, cannot pass through cell membranes hence,The bacteria should be made competent to accept the DNA molecules.
(1) competency is the ability of a cell to take a foreign DNA.
(2) Methods to make a cell competent are as follows.
(a) chemical method:-
* in this method, The cell is treated with a specific concentration of a divalent cation such as calcium to increase pore size in cell wall.
* The cells are then incubated with Recombinant DNA on ice followed by placing them briefly at 42 degree Celsius and then putting it back on ice. This is called heat shock treatment because of this permeability of bacterial cell increases.
* This enables the bacteria to take up the Recombinant DNA.
(b) Physical method
* In this method are Recombinant DNA is it directly injected into the nucleus of an animal cell by microinjection method.
* In plant cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA.This method is called as biolistic for gene gun method.
* this and pathogen vectors. These when allowed to infect the cell transfer the Recombinant DNA into the host.
These are very important topics for NEET-UG
* other related notes of biology
1-https://mbbsprep.blogspot.com/2020/03/key-tools-of-genetic-engineering.html?m=1
2-https://mbbsprep.blogspot.com/2020/03/principles-of-biotechnology-and.html?m=1
3- https://mbbsprep.blogspot.com/2020/03/adolescence-and-alcohol-drug-abuse.html?m=1
https://mbbsprep.blogspot.com/
- the DNA molecule that can carry a foreign DNA segment and replicate inside. The host cell is called a vector the host cell.
(1) the vectors used in Recombinant DNA technology can be
(a) plasmids autonomously replicating circular extrachromosomal DNA.
(b) bacteriophages virus infecting bacteria.
(c) cosmids hybrid vectors drive from plasma cell which contain cos site of Lambda phage
(2) copy number can be defined as the number of copies of vectors present in a Cell So, in rDNA technology is used in vectors are selected which have high copy number it can vary from 1- 100 or more than 100 copies per cell .
bacteriophages have hi number per cells with their copy number is also High in genome.
plasmids have Only one or two copies per cell.
(3) if an Alien piece of DNA is linked with bacteriophage of plasmid. DNA its number can be multiplied equal to the copy number of the plasmids or bacteriophage.
(4) present day vectors help in easy linking of foreign DNA and selection of recombinants from non Recombinant.
 |
| NEET-UG notes |
FEATURES Required To facilitate cloning into vector :-
(a) origin of replication (ori) is a sequence from where replication starts.
* Any piece of DNA when linked to this sequence can be made to replicate within the host cells.
* The sequence is also responsible for controlling the copy number of the linked DNA.
* So if one wants to recover many copies of target DNA it should be cloned in a vector whose Origins support High copy number.
(b) selectable marker helps in identifying and eliminating non transformants and selectivity permitting the growth of the Transformants.
* Transformation is a process through which the piece of the DNA is introduced in the host bacterium. Those host cells which have got Recombinant DNA are known as transformants,while those have not got the Recombinant DNA known as non-transformants.
* The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc are some useful . Selectable markers for E.coli.
* The normal E.coli cells do not carry resistance against any of these antibiotics.
 |
| E.coli cloning vectors pBR322 showing restriction sites |
(c) cloning sites are required to link the alien DNA with the vector. It contains restriction sites.
* The vector requires very few or single recognition sites for the commonly used restriction enzymes.
* The presence of more than one recognition sites within the vector will generate several fragments leads to complication in gene cloning. So one recognition site for an enzyme is preferred.
* Ligation of alien DNA is carried out at a restriction site presents in one of the two antibiotic resistance genes, for example, ligating a foreign DNA at the Bam HI site of tetracycline resistance clean in the vector pBR322.
* The Recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA but it still can be selected out from non Recombinant Once by plating in the Transformants on ampicillin containing medium.
* The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline.
* The Recombinant will grow in ampicillin containing medium but not on the containing tetracycline because in Recombinant tetracycline antibiotic resistance gene is the disturbed.
* The non Recombinant will grow on the medium containing both the antibiotic because both antibiotic resistance genes are functional.
* In this example one antibiotic resistance gene helps in selecting the transformants whereas the other antibiotic resistance genes get inactivated due to insertion of alien DNA and helps in selection of recombinants.
* Selection of recombinants due to inactivation of antibiotic is cumbersome procedure, because it requires simultaneous plating on two plates having different antibiotics. So alternative selectable markers are developed available which differentiate recombinants from non recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
* In this method, a recombinant DNA is inserted within the coding sequence of an enzyme Beta-galactosidase. So, if genes of interest gets incorporated,
Beta - galactosidase gene will be disturbed.
* This results in inactivation of the enzyme Beta-galactosidase (insertional inactivation).
* The bacterial colonies whose plasmids do not have an insert to produce blue colour when grown on a chromogenic substrate because of the presence of Beta galactosidase enzyme the presence of an insert in bacterial colonies does not produce any colour due to insertional inactivation of gene producing beta-galactosidase enzyme.
(d) Vectors for cloning genes in plants and animals several vectors are used to clone genes of interest in plants and animals.
* In plants, The Tumor inducing plasmids of agrobacterium tumefaciens is used as a cloning vector. It is known as Natural engineer of plants.
* Agrobacterium tumefaciens is a pathogen of several dicot plants.
* The tumor inducing plasmids of this bacteria has now been modified into a cloning vector which is no more pathogenic to the plants. It delivers a piece of DNA known as T-DNA in the Ti-plasmid which transforms normal plant cells into tumors cells to produce Chemicals required by the pathogens.
* Retrovirus, adenovirus, papillomavirus are also used as cloning vectors in animals because of their ability to transform normal size into cancerous cells.
Competent host organisms :-
- It is required because DNA being a hydrophilic molecule, cannot pass through cell membranes hence,The bacteria should be made competent to accept the DNA molecules.
(1) competency is the ability of a cell to take a foreign DNA.
(2) Methods to make a cell competent are as follows.
(a) chemical method:-
* in this method, The cell is treated with a specific concentration of a divalent cation such as calcium to increase pore size in cell wall.
* The cells are then incubated with Recombinant DNA on ice followed by placing them briefly at 42 degree Celsius and then putting it back on ice. This is called heat shock treatment because of this permeability of bacterial cell increases.
* This enables the bacteria to take up the Recombinant DNA.
(b) Physical method
* In this method are Recombinant DNA is it directly injected into the nucleus of an animal cell by microinjection method.
* In plant cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA.This method is called as biolistic for gene gun method.
* this and pathogen vectors. These when allowed to infect the cell transfer the Recombinant DNA into the host.
These are very important topics for NEET-UG
* other related notes of biology
1-https://mbbsprep.blogspot.com/2020/03/key-tools-of-genetic-engineering.html?m=1
2-https://mbbsprep.blogspot.com/2020/03/principles-of-biotechnology-and.html?m=1
3- https://mbbsprep.blogspot.com/2020/03/adolescence-and-alcohol-drug-abuse.html?m=1
https://mbbsprep.blogspot.com/
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