Abstract: Genotyping by gel electrophoresis can be a long and cumbersome task. Here, we present an alternative method for streamlining high-throughput fragment analyses. A segregating line of Arabidopsis thaliana LOH1 knockout seeds was grown and harvested at three weeks for genomic DNA extraction. The DNA was amplified with a three primer PCR method to screen for mutations. To determine the genotype, the PCR products were analyzed using the ZAG DNA Analyzer system with the ZAG 110 dsDNA kit (35-5000 bp). With this system, 96 samples were separated simultaneously using parallel capillary electrophoresis, and the results analyzed using ProSize data analysis software. The resulting digital gel image provided easy visualization of the results, with the wildtype sample displaying a large band at approximately 1,200 bp, the homozygous mutants a smaller band at approximately 500 bp, and the heterozygous plants showing both bands. Advanced options provide unbiased analysis of sizing and can flag samples based on user-defined criteria. Using these options, we created flags for the presence and/or absence of a band at each of the size ranges (1100 +/- 150 and 500 +/- 50). The results were exported in a table indicting the genotype of each sample, allowing for sample calls to be made without human error. The exported results matched the calls that were made by interpretation of the gel images. This high-throughput method of genotyping analysis with the ZAG DNA Analyzer system can easily be applied to other model organisms, including maize, mice, and zebrafish.Summary: A high-throughput genotyping workflow using parallel capillary electrophoresis
Ask the author a question about this poster.
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
Improving the Chemical Synthesis and Performance of Extended-Length Guide RNAs for Genome Editing and Control of Gene Expression
Laurakay Bruhn, Bo Curry, Rob Kaiser, Ben Lunstad, Ryan McCaffrey, Joel Myerson, Subhadeep Roy, Daniel Ryan, Israel Steinfeld, David Taussig, Justin Townsend, Suhani Thakker, Doug Dellinger
Modulating the Activity of CRISPR-Cas with Chemical Modifications in Single-Guide RNAs
Daniel Ryan, David Taussig, Suhani Thakker, Israel Steinfeld, Ben Lunstad, Rob Kaiser, Ryan McCaffrey, Justin Townsend, Patrick Chaffey, Bo Curry, Doug Dellinger, Laurakay Bruhn
The Reasons for Choosing Our Cancer Panels - CD Genomics
Build A Gene Mutation Panel with Non-NGS Technologies - CD Genomics
Why Cancer Panels Stand Out for Cancer Research