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.
Please join StudyMode to read the full document