TILLING: VERSATILE REVERSE GENETIC TOOL
Navgeet1, Balraj Singh Gill2, Arvind Negi3*, Shashi Shekhar Anand2
1Institute of Himalayan Bioresource and Technology (CSIR-IHBT), Palampur (HP)
2Centre for Biosciences Central University of Punjab, Bathinda (Punjab)
3Centre for Chemical and Pharmaceutical Sciences, Central University of Punjab, Bathinda (Punjab)
Recent years have been witnessedfor exhaustive genome sequencing, nourishing a cause to track the mutation and its consequences at the phenotypic level. Butreliabilityof these genomic studiesis the critical issuewhich is still unaddressed and not properly understood. Moreover via TILLING techniques (reverse genetic tool), can be valuable in evaluation of these studies to much extent. Usually TILLING account on a specific gene mutation in order to observe the extent of the functionality of that particular gene at morphological level. This review compiles the literature pertaining to the art of tilling in the evaluation of genomic studies and majorly in concern of functionalgenomics. Moreover this review also covers the example of the most common species so as to buildbetter understanding of the concept behind this technique of reversegeneticsand can be fruitfully applied in extractingthe ethano-botanical and therapeuticeffect of various medicinal plants.
REFERENCE ID: PHARMATUTOR-ART-2094
PharmaTutor (ISSN: 2347 - 7881)
Volume 2, Issue 1
Received On: 23/012/2014; Accepted On: 27/12/2014; Published On: 15/01/2014
How to cite this article: A Negi, Navgeet, BS Gill, SS Anand, Tilling: Versatile Reverse Genetic Tool, PharmaTutor, 2014, 2(1), 26-32
Exhaustive techniques exploration decodes the genome sequencing. Mostconcern belongs to DNA sequencing, BLAST searching and other Bioinformatics toolswhich retrieve the genetic information andcan be inferred in terms of the phenotype level of an organism. But interpretation at phenotypic level is not an easy task andhence it posesa challengingmenace to the new discoveries in genomics. To encounter this challenging situation most often implicate technique, RNAi also possessescertain shortcomings e.g.Delivering siRNAs to target loci . In persuing to overcomethis shortcoming, TILLING (Targeting Induced Local Lesions in Genomes)was introduced in the early 21st centurywhere it allow researchers to direct an identification of mutations in a specific gene. Initially it was implicated onaplant model, “Arabidopsis thaliana”, afterwards a large list of various plants which can be useful for pharmaceutical and phytochemical industry as via this technique they directed the gene regulated expression of a particular phytoconstituent. Meantime, this technique can increase the yield of the phytoconstituent or also can increase the adaptability of plant in the stress conditions.
Moreover since it’s discovering TILLING has seen considerably transformations and derivatizations which take its application into a new level. Now-a-days it isbecoming a conventionaltechnique in a field of reverse genetics which is successfully implicated oncorn, wheat, rice, soybean, maize, tomato and lettuce . It works on a very simple principle that mutations can be inducedviamutagens. Moreover ionizing radiation and certain chemicals can cause genes to mutate and made it possible to perform genetic studies that were not feasible initially when spontaneous mutations were only available!Afterwards the concerning principle was comprehensively implicated for analyzing gene function in order to understandthe pheno-genotyperelationship of higher organisms. Meanwhile alkylating agents which cause point mutations, valuedas their induced mutations brought altered and truncated protein products which can be helpfulin precise mapping of gene and protein function.
With the recent expansion of sequence databanks, locus-to-phenotype reverses genetic strategies sufficiently overcome the limitations of phenotypic screens for functional analysis. Even the retrieved sequence alone is sufficient to deduce its function viaBioinformatics comparative tools.
Most common methods for producing a reduction-of-function mutations are antisense RNA suppressionand insertional mutagenesiswhich are currently leading diagnostic tool of functional genomics. As these techniques highly rely on Agrobacterium T-DNA vectors for transmission or on an endogenous tagging system, their usefulness as general reverse genetics methods is limited to very few plant species. However, these techniques produce a very limited range of allele types.Meantime, lots of sequencingof Arabidopsis and other organismsurge a call to develop a genome-scale reverse genetic tools which are versatile, automated and capable of creating the wide range of mutant alleles that are needed for functional analysis .
Mechanism: In this method, chemically assisted mutagenesis is done to cause point mutation on the targeted loci of a sensitive DNA. TILLING process includesthe formation of DNA hetroduplexes followed by amplification with PCR.With amplification,mismatch bubble formation take place between two strands which further cleaved by nucleases. Mismatch plays cardinal role in TIILING and may be induced by different means,seein fig. 1 and fig. 2.
Fig. 1.Concerning steps of mechanism.
Fig. 2. Different steps involved in TILLING process.
Edges of TILLING Over the Conventional Techniques
As compared with other conventional transgenic techniques, TILLING always placed aspecific edgein the identification of numerous mutations within the targeted region of the genome where region leads to phenotypes and hence promote the Geno-phenotype studies. In transformation, RNAi although able to provide genetic characterization, but it encountered certain shortcoming during knockout in a series. While TILLING incomparisonto RNAi affordconsistent analysis by forming a library.
It yields a traditional allelic series of point mutations.
Very essential for phenotypic analysis of sub-lethal alleles.
Since it uses chemical mutagenesis virtually all genes can be targeted by screening few individuals.
Versatile, hence can be applied to virtually any organism.
It offers high-throughput screening.
Independent of genome size, reproductive system or generation time.
Assist in detection single base paired polymorphism.
Critical incidences are also available where TILLING was most favored and provide significant results
Chemical mutagenesis was applied to soybeanfollowed by screening for mutations in a target of interest using a strategy known as TILLING. Later on TILLING was employed to 4 mutagenized soybean populations, 3 of which were treated with ethyl methane sulfonate (EMS) andremaining one with N-nitroso-N-methylurea (NMU).
The mutation was discovered in mutagenized soybean populations. Truncation, missense and silent mutations were observed. TILLING was done in following steps:
Mutagenesis and DNA preparation.
The primer was designed using CODDLe (Codon Optimized to Deliver Deleterious lesions).
High throughput TILLING
LICOR 4200 or 4300 gel images were analyzed using gel buddy .
Non-denaturing polyacrylamide gel set-up was employed as an alternative for LICOR screens, to make it easy and cost effective.
· Generation of EMS mutagenized population.
EMS = 0.7-0.75%
Germination rates for EMS treated seeds were ~50-60%
· Development of genome specific primers: primers were designed complementary to intron sequences flanking the target exons and positioned approximately 200bp from sequence of interest.
· Gene libraries were characterized using
SBEIIa, SBEIIb – starch branching enzyme II genes.
WKS1, WKS2 – wheat kinase start genes
· Wheat TILLING platform using non-denaturing polyacrylamide detection method.
CODDLe program was used to predict the effect of EMS mutations.
Hexaploid - 40% missense mutations
Tetraploid – 28%missense
· Comparison between LICOR and non-denaturing polyacrylamide detection methods was done.
LICOR – 1 mutation/40kb
Non-denaturing polacrylamide – 1 mutation/41.5 kB.
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