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Indel Detection by Amplicon Analysis (IDAA)
Indel Detection by Amplicon Analysis (IDAA) is an advanced method designed for pinpointing insertions and deletions (indels) resulting from precise gene editing. BioVenic is dedicated to providing IDAA services for genome modification validation in the field of precision animal breeding.
What is IDAA?
IDAA is an innovative approach, merging a straightforward amplicon labeling method with high-throughput DNA fragment analysis through automated Capillary Electrophoresis. It excels in detecting indels, even in low-efficiency cell pools and individual sorted cells. It's ideal for high-throughput detection, pinpointing single-base indel events, assessing targeting tool efficiency, and evaluating off-target events at specific loci.
Workflow of IDAA
In precise gene targeting, double-stranded breaks are intentionally induced at the target site, often leading to the introduction of insertion or deletion mutations (indels) via nonhomologous end-joining (NHEJ) repair. To identify and analyze these indels, the Tri-primer PCR method is employed. This technique involves three primers: two specifics to the target site (one for the forward strand, F, and one for the reverse strand, R), and a third universal primer labeled with a fluorescent marker (5'-FAM labeled primer, FamF). This universal primer's fluorescence allows for precise labeling of resulting amplicons during PCR. Fluorescently labeled amplicons, containing indels, are then separated by size and fluorescence intensity in fragment analysis. This data is plotted with axes representing fluorescence intensity (FI) and amplicon size (in base pairs), facilitating indel identification and characterization in the target region.
Fig.1 Schematic diagram of the IDAA assay. (BioVenic Original)
High-Throughput IDAA Assay for Validation
BioVenic provides high-thoughput IDAA assay for animal gene editing validation, which allows for high-throughput, precise detection, and characterization of indels in the target region.
Target-specific primer design
Tri-primer PCR incorporating a fluorescently labeled primer
Electrophoresis
Fragment analysis
Our Advantages
Our IDAA services are here to support you in precision animal breeding whether you are working on gene editing, TALENs, or other precise genome editing techniques.
We employ the IDAA techniques to ensure accurate and sensitive detection of indels as small as a single nucleotide.
Our IDAA services can be tailored to your specific requirements, accommodating a wide range of target nucleic acid sequences, sample sizes, and data analysis needs.
Our team of molecular biologists and geneticists is highly skilled in using IDAA to uncover even the most challenging indels.
BioVenic is dedicated to revolutionizing animal breeding through advanced IDAA technologies. You gain access to a reliable and efficient solution for indel detection, allowing you to focus on advancing your animal research goals. Please don't hesitate contact us to learn more.
References
- Yang, Zhang, et al. "Fast and sensitive detection of indels induced by precise gene targeting." Nucleic acids research 43.9 (2015): e59-e59
- Lonowski, Lindsey A., et al. "Genome editing using FACS enrichment of nuclease-expressing cells and indel detection by amplicon analysis." Nature protocols 12.3 (2017): 581-603.