TILLING, and EcoTILLING have been widely used since 2000 to detect Single Nucleotide Polymorphisms (SNPs) in organisms ranging from Arabidopsis to zebra fish. The test samples may be experimentally mutagenized (ethylmethanesulfonate, radiation, etc.), from natural populations or derived from tumors or diseased tissues.
The linked TILLING analysis module uses data analyzed with GeneMarker® software's accurate Size Calling algorithms and customized Run Wizard provide rapid analysis of raw data from MegaBASE®, Beckman-Coulter® or ABI®PRISM capillary electrophoresis or slab gels (such as LICOR® platforms)analyzed with JelMarker® software.
Briefly, the genes of interest are identified with gene‐specific primers and PCR amplified. The amplicon's primers are labeled with two fluorescent dyes - the forward is often labeled with FAM‐blue and the reverse primer is often labeled with HEX‐green. The samples are mixed so heteroduplexes can be formed. The hybridized fragments are cleaved at the heteroduplex site by CEL I or Surveyor™ Nuclease, generating multiple pairs of fragments of complementary length and dye color. The denatured samples can be run through gel electrophoresis or mixed with an internal size standard and run through capillary electrophoresis. SNPs will yield two peaks of different color and the sum of the sizes will equal the amplicon length.
GeneMarker software TILLING Analysis
The top panel shows the Synthetic Control Sample obtained from the median intensity after peak alignment. The middle panel shows sample 79. The bottom panel shows the Mutation Chart, generated by subtracting the reference from the sample, identifying individual variations. A blue peak at 205.2bps and a green peak at 848.8bps have been automatically identified. The original amplicon size is 1049bps.
When using GeneMarker software's Tilling Application module, the size standard peaks are identified and all sample peaks are aligned. The peaks are smoothed, baseline is subtracted, and lane intensities are normalized. Low quality data is automatically rejected. A synthetic reference trace (Synthetic Control Sample) is constructed using median peak intensities from all of the high quality traces. This reference is subtracted from each sample trace generating a Mutation Chart that automatically identifies the sample's variations as shown in figure with the amplicon length of 1049bps.
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