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MultiFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction.

Cassidy PetreeGuarav K Varshney
Published in: Scientific reports (2020)
Powerful and simple, RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish, and can be used to target multiple genes simultaneously. One of the challenges associated with the large scale application of this technique to zebrafish is the lack of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing - a robust and cost-effective method to genotype multiple targets in a single reaction. Our approach can identify indels in up to four targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis-based sequencing equipment.
Keyphrases
  • crispr cas
  • genome editing
  • genome wide
  • high throughput
  • high resolution
  • capillary electrophoresis
  • dna methylation
  • single cell
  • copy number
  • gene expression
  • quantum dots
  • multidrug resistant
  • genetic diversity