Mutations are genetic changes or modifications caused by chemical and physical mutagens. Mutations can results from modification of a single base or few bases. However this can result in change or modification of a phenotypic character which can be used to recognize them. This feature is widely used in DNA recombinant technology. Plasmid vectors carry genes for drug resistance, toxin production which can be used to distinguish recombinants. When genes of interest are inserted into the plasmid, the reading frame for the marker genes can be altered. This results in mutants who can be identified using special chemicals/ media.
AMES test is one such method to identify mutants of Salmonella typhimurium that cannot produce Histidine. This mutant stain can be cultured only when Histidine is present in the basic medium. This is the standard culture used for testing chemical mutagens. The chemical mutagen is loaded into a well in the centre of a culture plate of inoculated with Salmonella typhimurium in a medium lacking Histidine. The chemical diffuses into the medium. A growth indicates that the chemical has induced a mutation in Histidine- stain converting it to Histidine +. Depending on the position of the colony relative to the well containing the chemical, the degree of resistance varies. Colonies growing closer to the well are quite resistant and colonies growing at the periphery indicate that the chemical even at a low concentration can induce mutation. This test has its application in pharmaceutical industry to test the effect of drugs; whether it’s a mutagen or not.
In replica plating method to screen mutants, the organism is subjected to radiation or exposed to chemical to induce the mutation. The mutants can be identified by a reduction in the colony size or change in pigmentation etc. Sub culture is made by replica plating the master culture. The sub cultured plate is subjected to mutation and incubated for growth. Then the plate subjected to mutation is replica plated onto a fresh medium and incubated to observe phenotypic variations. This method can be used to identify the dosage of radiation or chemicals required to cause mutations. If this is repeated with different dosage levels, the finally left colony will be having most resistant cells for the particular mutagen.
Gradient method is used to study the effect of chemical mutagens on bacteria. A medium containing two different concentrations are prepared separately and poured over the same plate in a slanting position. First the medium with lower concentration of the chemical is poured onto the plate in a slanting position and allowed to solidify. Then the medium with higher concentration of the chemical is poured onto the plate. The plate is inoculated and observed for growth. This is repeated till there is no growth at higher concentration to identify the effective concentration.
Blue white selection is a widely used method in screening recombinants in cloning. This is based on the gene product of lac z gene. The plasmid vectors contain this gene which produces β galactosidase enzyme. When a gene is inserted close to lac z gene, the reading frame will be distorted and the gene is inactivated. So the transformed cells will not produce this enzyme and are called competent cells. After the recombination, the bacterial cells are grown in a medium containing X gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) and IPTG (Isopropyl β-D-1-thiogalactopyranoside). IPTG acts as the inducer for lac z gene and enhance the production of β galactosidase. When it is produced, combines with X gal to form a blue colour complex called 5,5'-dibromo-4,4'-dichloro-indigo which is insoluble. The transformed colonies will appear white in colour and non- transformed cells will appear blue in colour. This method is also called as insertional inactivation of lac z gene.
Hybridization techniques are widely used to identify recombinants. This is based on the ability of nucleic acids hybridize with complementary DNA. The transformed cells are transferred on to a nitrocellulose membrane which is subjected to cell lysis. The double stranded DNA is converted to single stranded DNA and immobilized on the membrane. Then it is treated with radiolabelled probes complementary to target DNA. If the desired DNA is present, the probes will be hybridized which can be detected by autoradiography.
Apart from these methods, immunochemical methods are used to detect protein products to screen recombinants.
AMES test is one such method to identify mutants of Salmonella typhimurium that cannot produce Histidine. This mutant stain can be cultured only when Histidine is present in the basic medium. This is the standard culture used for testing chemical mutagens. The chemical mutagen is loaded into a well in the centre of a culture plate of inoculated with Salmonella typhimurium in a medium lacking Histidine. The chemical diffuses into the medium. A growth indicates that the chemical has induced a mutation in Histidine- stain converting it to Histidine +. Depending on the position of the colony relative to the well containing the chemical, the degree of resistance varies. Colonies growing closer to the well are quite resistant and colonies growing at the periphery indicate that the chemical even at a low concentration can induce mutation. This test has its application in pharmaceutical industry to test the effect of drugs; whether it’s a mutagen or not.
In replica plating method to screen mutants, the organism is subjected to radiation or exposed to chemical to induce the mutation. The mutants can be identified by a reduction in the colony size or change in pigmentation etc. Sub culture is made by replica plating the master culture. The sub cultured plate is subjected to mutation and incubated for growth. Then the plate subjected to mutation is replica plated onto a fresh medium and incubated to observe phenotypic variations. This method can be used to identify the dosage of radiation or chemicals required to cause mutations. If this is repeated with different dosage levels, the finally left colony will be having most resistant cells for the particular mutagen.
Gradient method is used to study the effect of chemical mutagens on bacteria. A medium containing two different concentrations are prepared separately and poured over the same plate in a slanting position. First the medium with lower concentration of the chemical is poured onto the plate in a slanting position and allowed to solidify. Then the medium with higher concentration of the chemical is poured onto the plate. The plate is inoculated and observed for growth. This is repeated till there is no growth at higher concentration to identify the effective concentration.
Blue white selection is a widely used method in screening recombinants in cloning. This is based on the gene product of lac z gene. The plasmid vectors contain this gene which produces β galactosidase enzyme. When a gene is inserted close to lac z gene, the reading frame will be distorted and the gene is inactivated. So the transformed cells will not produce this enzyme and are called competent cells. After the recombination, the bacterial cells are grown in a medium containing X gal (5-bromo-4-chloro-indolyl-β-D-galactopyranoside) and IPTG (Isopropyl β-D-1-thiogalactopyranoside). IPTG acts as the inducer for lac z gene and enhance the production of β galactosidase. When it is produced, combines with X gal to form a blue colour complex called 5,5'-dibromo-4,4'-dichloro-indigo which is insoluble. The transformed colonies will appear white in colour and non- transformed cells will appear blue in colour. This method is also called as insertional inactivation of lac z gene.
Hybridization techniques are widely used to identify recombinants. This is based on the ability of nucleic acids hybridize with complementary DNA. The transformed cells are transferred on to a nitrocellulose membrane which is subjected to cell lysis. The double stranded DNA is converted to single stranded DNA and immobilized on the membrane. Then it is treated with radiolabelled probes complementary to target DNA. If the desired DNA is present, the probes will be hybridized which can be detected by autoradiography.
Apart from these methods, immunochemical methods are used to detect protein products to screen recombinants.
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