Tilling [targeting induce local lesions in genome

Tilling [targeting induce local lesions in genome

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  TILLING [TARGETING INDUCE LOCALLESIONS IN GENOME] Presented By: Prashansa Tayal , M.Sc Biotechnology [3rd Sem] Chaudhary Charan Singh University, Meerut, U.P. India. Supervisor : Dr. Pradip Kumar Department of Biotechnology C.C.S. University, Meerut
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  TILLING [Targeting Induce LocalLesions in Genome] For Gene Function GENE MUTATION PHENOTYPIC CHANGE IN MUTATED ORGANISM GENE SEQUENCE FORWARD GENETIC APPROACH [ PHENOTYPE GENE] LARGE MUTATED POPULATION HAVE BEEN CREATED SERVED FOR THE IDENTIFICATION OF GENE UNDERLYING THE CHANGE IN PHENOTYPE SEQUENCE OF THE GENE CHANGE IN ALTERED PENOTYPE CAN BE ISOLATED BY MAP BASED CLONNING TIME CONSUMING & LABOUR INTENSIVE
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  REVERSE GENETIC APPROACH[ GENE SEQUENCE PHENOTYPE] ALTERATION OF GENE STRUCTURAL ACTIVITY ANALYSIS OF ASSOCIATED CHANGE IN PLANT PHENOTYPE Examples :- • INSERTIONAL MUTAGENESIS WITH T-DNA • TRANSPOSONS TAGGING • GENE SILENCING USING RNA INTERFERENCE • TILLING Continued....
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  KUROWSKA , M[2011] et al
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  TILLING • Alternative toinsertional mutagenesis. • Take advantage of classical mutagenesis, sequence availability & high throughput for screening nucleotide polymorphism in a target sequence. • Applied to any plant species, regardless of its genome size, ploidy level/ method of propagation. • Much smaller populations are required to reach saturation mutagenesis using TILLING as compared to T- DNA mutagenesis. • Chemical mutagens provide a high frequency of point mutation, distributed randomly in the genome.Create a spectrum of mutationGain of function alleles -Loss of functions alleles - Hypomorphic alleles - -Missense changes -Truncation -Mutation in splice junction sequences
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  • In contrastto insertional mutagenesis that generates mostly gene knockouts, using TILLING, it is possible to induce a series of alleles in a targeted locus. • Does not required transformation processes like RNAi technology & insertional mutagenesis. • Non-GMO technology. • Initially developed as a discovery platform for functional genomics, but it soon become a valuable tool in crop breeding as an alternative to the transgenic approach. TILLING Continued….
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  GENERAL PROTOCOL 1. Creationof mutated population – • Chemical mutagenesis [EMS mainly] • Development of M1 and M2 generations • DNA extraction from individual M2 plants • Creation of DNA pools of 5-8 M2 plants • Setting up an M3 seed bank. 2. Detection of mutation in a targeted sequence- • PCR amplification of the targeted DNA segment using pooled DNA as a template. • Detection of mutations [ cleavage by specific endonuclease] • Identification of the individual M2 plant carrying the mutation. • Sequencing the target gene segment to confirm the mutation & to determine the type of nucleotide change. 3. Analysis of the mutant phenotype
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  KUROWSKA , M[2011] et al
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  Major components ofTILLING Procedures 1. Identifying Experimental material – Seeds mainly but other parts of plants can be used. 2. Determining the type & conc. of the mutagen - Mainly EMS creates G : C to A : T transitions. 3. Creating mutagenized population – M2 population 4. DNA isolation and pooling – from each M2 plant. 5. Primer design and PCR amplification – design a set of primers which are specific to amplify only the gene of interest. - PCR amplification is followed by the heteroduplex formation step where the products of the PCR are first denatured followed by slow annealing to facilitate the formation of heteroduplex molecules. 6. Mutation detection – by S2 nuclease family [CEL 1, Mung bean nuclease] 7. Validation of mutation by sequencing & crossing
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  REFERENCES • Kurowska, M.,Daszkowska- Golec, A., Gruszka, D., Marzec, M., Szurman, M., Szarejko, I., & Maluszynski, M. [2011] . TILLING a shortcut in functional genomics. Journal of applied genetics, 52(4),37. • Tadele, Z., [2016]. Mutagenesis and TILLING to dissect gene function in plants. Current genomics,17(6), 499- 508.
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  Any questions?