Methods for clone identification
- 1. PhD in Biotechnology/Microbiology/Biochemistry UNIT- RDT Topic: Methods for clone identification By Dr. Vijay Kumar Assistant Professor Department of Biosciences, Swami Rama Himalayan University
- 2. Methods for clone identification Hybridization probing Complementary nucleic acid strands hybridize to each other Colony and plaque hybridization probing Labeling with a radioactive marker Non-radioactive labeling Examples of the practical use of hybridization probing Abundancy probing to analyze a cDNA library Heterologous probing allows related genes to be identified Southern hybridization enables a specific restriction fragment containing a gene to be identified Identification methods based on detection of the translation product of the cloned gene immunological screening OUTLINE OF THE TOPIC
- 3. Learning objectives At the end of this learning session a student of M.Sc. Microbiology/Biotechnology –Semester-II should be able to: Learn about major methods used to identify clones Hybridization methods Probe labelling methods Identification of clone by immunological screening
- 4. Introduction Once a suitable library has been prepared, a number of procedures can be employed to attempt identification of the desired clone Few of these procedures are based on detection of the translation product of the cloned gene It is usually easier to identify directly the correct recombinant DNA molecule Can be achieved by the important technique of hybridization and immunological screening
- 5. Complementary nucleic acid strands hybridize to each other Nucleic acid hybridization An unstable hybrid A stable hybrid Short non-complementary regions do not affect overall stability An unstable hybrid molecule formed between two non-homologous DNA strands. A stable hybrid formed between two complementary strands A DNA–RNA hybrid A DNA–RNA hybrid, such as may be formed between a gene and its transcript.
- 6. Colony and plaque hybridization probing Nitrocellulose/nylon membrane Alkali + protease Bacteria attached to membrane 80°C for 2 hours or ultraviolet irradiation DNA bound to the membrane OR Probe with labelled DNA Wash Apply X-ray film The resulting autoradiograph
- 7. Methods for labelling DNA Labelling by nick translation Labelling by end-filling Labelling by random priming Labeling with a radioactive marker DNA molecule is usually labeled by incorporating nucleotides that carry a radioactive isotope of phosphorus, 32P
- 8. Non-radioactive labeling Labelling with a biotinylated nucleotide DNA probe Biotin–dUTP Nick translation, end- filling or random priming Detect with avidin coupled to a fluorescent marker
- 9. Labelling with horseradish peroxidase
- 10. mRNA can be cloned as complementary DNA 1. First strand synthesis 4. Ligation into a vector 3. Second strand synthesis 5. Transform 2. RNA degradation
- 11. Probing within a library to identify an abundant clone
- 12. Oligonucleotide probes for genes whose translation products have been characterized Bind first nucleotide to the support
- 13. The use of a synthetic, end-labelled oligonucleotide to identify a clone of the yeast cytochrome c gene
- 15. Southern hybridization enables a specific restriction fragment containing a gene to be identified A long cloned DNA fragment may contain several genes in addition to the one in which we are interested. B = BamHI restriction site Southern hybridization.
- 16. Identification methods based on detection of the translation product of the cloned gene Antibodies. (a) Antibodies in the bloodstream bind to foreign molecules and help degrade them. (b) Purified antibodies can be obtained from a small volume of blood taken from a rabbit injected with the foreign protein.
- 17. Using a purified antibody to detect protein in recombinant colonies. Instead of labelled protein A, the antibody itself can be labelled, or alternatively a second labelled antibody which binds specifically to the primary antibody can be used.
- 18. Suggested readings • Brown, T. A. (1990). Gene cloning (pp. 2-6). Chapman and Hall. • Old, R. W., & Primrose, S. B. (1981). Principles of gene manipulation: an introduction to genetic engineering (Vol. 2). Univ of California Press.