Differential gene profiling methods
- 1. Differential gene profiling methods Sonam Yadav M.Sc. Botany 4th Sem.
- 2. Differential gene profiling • Gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. • These profiles can distinguish between cells that are actively dividing, or show how the cells react to a particular treatment. Many experiments of this sort measure an entire genome simultaneously ; every gene that is present in a particular cell.
- 3. Differential gene expression • Through the process of differential gene expression, the activation of different genes within a cell that define its purpose, each cell expresses only those genes which it needs. However, the extra genes are not destroyed, but continue to be stored within the nucleus of the cell.
- 4. Methods of gene expression
- 5. Why is differential gene profiling important? • Gene expression profiling measures which genes are being expressed in a cell at any given moment • This method can measure thousands of genes at a time; some experiments can measure the entire genome at once.
- 6. Eg.-Differential gene expression in leaf tissues between mutant and wild-type genotypes response to late leaf spot in peanut (Arachis hypogaea L.)
- 8. • Analysis of the differential gene and protein expression profile of the rolled leaf mutant of transgenic rice (Oryza sativa L.)
- 9. TYPES 1. Differential display 2. Subtractive hybridization
- 10. DIFFERENTIAL DISPLAY • Differential display is a powerful technique for detecting and quantitating changes in gene expression patterns between differently treated cells. • Fragments of those genes which are induced or suppressed can be identified and isolated for further analysis, with no prior knowledge of the sequences involved. The technique is PCR based, and yields results in only 1 - 3 days. • DDRT-PCR was developed by Liang and Pardee.
- 12. •Differential display (also referred to as DDRT-PCR or DD- PCR) is a laboratory technique that allows a researcher to compare and identify changes in gene expression at the mRNA level between two or more eukaryotic cell samples. •It was the most commonly used method to compare expression profiles of two eukaryotic cell samples in the 1990s. • By 2000, differential display was superseded by DNA microarray approaches.
- 13. POTENTIAL APPLICATIONS • DDRT-PCR has been used to answer many biological questions in mammalian and other biological systems. • RT-PCR to examine the expression of each FRO gene in different tissues of Arabidopsis ‘s root and in response to iron and copper limitation. • (FRO- Ferric reductase oxidase)
- 14. • This method is also used in the identification of differentially expressed genes in developing cotton fibre. • The multidimensional applications of DDRT PCR in study the differentially expressed gene under biotic and abiotic stress especially the salinity stress. Eg. In Rice (Oryza sativa) -Expression of SAMDC1 gene positively correlates with salt tolerance in rice.
- 15. SUBTRACTIVE HYBRIDIZATION • Subtractive hybridization is a technique for identifying and characterizing differences between two populations of nucleic acids. • It detects differences between the RNA in different cells ,tissues,organisms,or sexes under normal conditions,or during different growth phases,after various treatments (i.e, Hormone application,heat shock) or in diseased(or mutant) versus healthy(or wild-type) cells.
- 16. • The two population are hybridized with a driver to tester ratio of at least 10:1. • Because of the large excess of driver molecules, tester sequences are more likely to from driver-tester hybrids than double standard tester. • Only the sequences in common between the tester and the driver hybridize , however, leaving the remaining tester sequences either single standard or forming tester-tester sequences.
- 17. • The driver-tester ,double-standard and any single standard driver molecules are subsequently removed(the “subtractive”step, leaving only tester molecules enriched for sequences not found in the driver. T D T TT TD DD D × × × ×
- 18. PROCESS Choosing material for isolating tester and driver nucleic acid. producing tester and driver. hybridizing them Removing driver-tester hybrids and excess driver(subtraction) Isolating of the complete seq. of remaining target nucleic acid.
- 20. Applications • simplified, fast and reliable method for generating subtracted probes or subtracted cDNA libraries. • Only small amounts of sample/mRNA are required • Magnetic handling minimizes losses at each step • Specific mRNAs are highly enriched • Magnetic handling enables simple and rapid buffer changes to optimize conditions for hybridization and specific enzymatic reactions.
- 21. REFRENCES • www.google.com,wikipedia. • Subrahmanyam P, Rao VR, McDonald D, Moss JP, Gibbons RW. Origins of resistances to rust and late leaf spot in peanut (Arachis hypogaea, Fabaceae). Economic Botany. 1989; 43(4):444–455. • pandey A; Mann, M (15 June 2000). "Proteomics to study genes and genomes". Nature. 405 (6788): 837–46. . • Reece, Richard J. (2004). Analysis of genes and genomes. Chichester: Wiley. ISBN 978-0-470-84380-2 • . • Galbraith, E. A.; Antonopoulos, D. A.; White, B. A. (2004). "Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: The rumen as a model". Environmental Microbiology. 6 (9): 928–937. doi:10.1111/j.1462- 2920.2004.00575.x. PMID 15305918. •