TILLiNG Detection

Reliable detection of TILLiNG induced mutations is crucial to interpreting results from this reverse genetics strategy.


What is TILLiNG?

Reverse genetics determines the function of genes of interest by analyzing the phenotypic effects of mutagenesis. In practice this involves the disruption of normal gene function with subsequent observation of any changes in phenotype. A particularly useful reverse genetics approach is Targeted Induced Local Lesions in Genomes (TILLiNG). Developed in Arabadopsis thaliana, TILLiNG was introduced in 2000.

Commonly used in plant genetics, TILLiNG provides researchers with a powerful tool for phenotypic investigation. The underlying strategy of TILLiNG is quite simple. Chemical mutagenesis is performed, introducing mutations that disrupt gene function. After mutagenesis, researchers take note of any changes in the expected phenotype. DNA is then extracted for use in a heteroduplex cleavage assay, allowing users to pinpoint what genes have been affected.

TILLiNG provides researchers with a powerful tool for exploring the relationship between phenotype and genotype. While an excellent method to identify mutated genes, and efficient fragment visualization strategy must be employed to take full advantage of TILLiNG.

A reverse genetics strategy commonly employed in agriculture, TILLiNG provides researchers with a powerful tool to probe the relationship between phenotype and genotype. TILLiNG has been used to elucidate the basis of desirable phenotypic traits in agriculturally significant traits that can then be selectively bred for or engineered into crops.

Efficient visualization of heteroduplex cleavage assay is a critical component of TILLiNG workflows. Poor methods of visualizing and quantifying heteroduplex cleavage assay results risk the development of workflow bottlenecks and a high error rate. TILLiNG workflows can be improved by incorporating high-throughput, high-accuracy DNA fragment analysis strategies. Notably, capillary electrophoresis offers exciting options for TILLiNG fragment analysis.

Accurate mutation detection and analysis are crucial to TILLiNG, enabling researchers to draw conclusions about what genes are involved in specific phenotypic traits. The enzyme kit, DNF-480 dsDNA Cleavage Kit, provides reliable and consistent detection of mutations via a robust heteroduplex cleavage assay.

Visualization of the assay results takes place on one of two specially designed gel kits for the Fragment Analyzer or the 5400 Fragment Analyzer: the DNF-910T Mutation Discovery 910 Gel Kit and the DNF-920T Mutation Discovery 920 Gel Kit. While these kits utilize the same ladder, the gel formulations confer higher resolution separations to different sizes of fragments. Should smaller amplicons be used in the heteroduplex cleavage assay, the DNF-910T Mutation Discovery 910 Gel Kit is recommended as it offers superior resolution for fragments below 1,000 bp. Similarly, if larger amplicons are used for the heteroduplex cleavage assay, the DNF-920T Mutation Discovery 920 Gel Kit provides enhanced separation for fragments between 1,000 bp and 3,000 bp.

Further simplifying mutation analysis with the Fragment Analyzer and the 5400 Fragment Analyzer, ProSize Data Analysis Software automatically processes data, indicating which sample wells have cleavage events for further investigation. Parent amplicons are marked in red, while cleavage products are in orange.

Figure 1. Electropherogram of a TILLiNG cleavage event. Parent amplicon shown in red, cleavage products are highlighted orange. Sample generated with the DNF-480 dsDNA Cleavage Kit. Separations performed with the DNF-910T Mutation Discovery 910 Gel Kit on a Fragment Analyzer equipped with a Short Capillary Array (33-55).

TILLiNG Mutation Detection Kits

The Fragment Analyzer and the 5400 Fragment Analyzer from Agilent - formerly Advanced Analytical (AATI) provide parallel capillary electrophoresis solutions to TILLiNG fragment analysis. With an enzyme kit and two kit options for TILLiNG fragment analysis, the instruments from Agilent - formerly AATI offer a superior solution for TILLiNG analysis.

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