One-pot DTECT enables rapid and efficient capture of genetic signatures for precision genome editing and clinical diagnostics.

The detection of genomic sequences and their alterations is crucial for basic research and clinical diagnostics. However, current methodologies are costly and time-consuming and require outsourcing sample preparation, processing, and analysis to genomic companies. Here, we establish One-pot DTECT, a platform that expedites the detection of genetic signatures, only requiring a short incubation of a PCR product in an optimized one-pot mixture. One-pot DTECT enables qualitative, quantitative, and visual detection of biologically relevant variants, such as cancer mutations, and nucleotide changes introduced by prime editing and base editing into cancer cells and human primary T cells. Notably, One-pot DTECT achieves quantification accuracy for targeted genetic signatures comparable with Sanger and next-generation sequencing. Furthermore, its effectiveness as a diagnostic platform is demonstrated by successfully detecting sickle cell variants in blood and saliva samples. Altogether, One-pot DTECT offers an efficient, versatile, adaptable, and cost-effective alternative to traditional methods for detecting genomic signatures.

A detection method for the capture of genomic signatures: From disease diagnosis to genome editing.

Variations in the genetic information originate from errors during DNA replication, error-prone repair of DNA damages, or genome editing. The most common approach to detect changes in DNA sequences employs sequencing technologies. However, they remain expensive and time-consuming, limiting their utility for routine laboratory experiments. We recently developed DinucleoTidE Signature CapTure (DTECT). DTECT is a marker-free and versatile detection method that captures targeted dinucleotide signatures resulting from the digestion of genomic amplicons by the type IIS restriction enzyme AcuI. Here, we describe the DTECT protocol to identify mutations introduced by CRISPR-based precision genome editing technologies or resulting from genetic variation. DTECT enables accurate detection of mutations using basic laboratory equipment and off-the-shelf reagents with qualitative or quantitative capture of signatures.