Contact:
sales@biotechnologyforums.com to feature here

Thread Rating:
  • 0 Vote(s) - 0 Average
  • 1
  • 2
  • 3
  • 4
  • 5
Screening Methods for Mutants/Recombinants in Recombinant Technology
#1
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.
Like Post Reply
#2
Additional Methods for Screening and Selection of Recombinants

Antibiotic resistance

This is one of the simplest selection methods. The plasmid of our interest should contain a specific gene for antibiotic resistance. For example, plasmid pBR322 contains the resistance for ampicillin and tetracycline. If the cells are successfully transformed (if they have taken up the plasmid), they will be resistant to both of these antibiotics, so other unwanted cells which do not contain our gene of interest can be easily eliminated simply by placing the cells on agar medium which contains either one or both of these antibiotics. This method works well also on the mammalian cells.

Insertional inactivation

Insertional inactivation helps us detect the target DNA because it disrupts the coding sequence once it has recombined. For example, if the target DNA gets inserted into the sequence coding for ampicillin resistance (because specific restriction enzyme cuts exactly at that position), it renders the cell which has successfully transformed sensitive to the ampicillin. What we can do next is grow cells in the culturing medium and wait for the colonies to form. We can then make a replica plate, expose it to the ampicillin and mark which colonies are sensitive, so we can work with them on the original plate.

Plaque morphology

There are some λ vectors (bacteriophage type of vectors) like λgt10 which encode the cI gene. The cI gene codes for the cI repressor proteins which allow phage lambda to transform the bacteria and start the lysogenic cell cycle. This basically means that the phage will live quietly in the cell, integrated into its genome (as a prophage) instead of multiplying itself, destroying the cell and exiting to attack the surrounding cells (lytic cycle). Plaques derived from the cI+ vectors will be turbid because some cells in the lysogenic cycle have survived. However, if the cI gene is inactivated during the process (due to the insertion of our fragment), the plaques formed will be clear and easily distinguishable from the turbid non-recombinants.
Like Post Reply
#3
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.

Prevention is better than cure. But there is no effective prevention of cancer. Surgery, radiotherapy and chemotherapy are some procedure to cure cancer. But they are not harmless and secured. Genetic engineering may be the latest solution of cancer prevention. Some research has been done to discover new path of cancer prevention through genetic engineering. Genetic changes caused by events such as mutation and agents such as viruses are recognized for research. Damaged genes are trying to recover in the prevention of cancer. If the genetic factors and processes which are responsible for cancer can be controlled in the primary stage, the further progression of cancer will be controlled. Those works must be done by genetic engineering. This genetic engineering ensures the protection of cancer cells from further mutation. Genetic engineering is done if cancer is affected by genetic problems. If carcinogens create cancer then preventive steps should be taken against carcinogens. Genetic engineering may be the best solution for cancer. But this matter is still under research. Our expectation may be fulfilled in the future.
We offer guaranteed success for online ccna training exam with help of latest network+ training courses. Good Luck
Like Post Reply
#4
Great article. Thanks.
Like Post Reply
  

Possibly Related Threads...
Thread
Author
  /  
Last Post



Users browsing this thread:
1 Guest(s)

Screening Methods for Mutants/Recombinants in Recombinant Technology00