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IDT’s rhAmpSeq™ technology is a PCR-based enrichment method for use on Illumina’s next generation sequencing platforms. The foundation of this product is IDT’s RNase H2 amplification technology. Upon hybridization of the primers to their specific target site, the RNase H2 enzyme cleaves the primer upstream of the RNA base. The cleavable, blocked primers are ideal for applications requiring large, multiplexed, PCR primer panels to help reduce the formation of primer dimers.
Applications for this technology include detection of genetic markers in agricultural samples, detection of mutational hot spots relevant to oncology, and evaluation of on- and off-target genomic editing by CRISPR-Cas9. Custom rhAmpSeq Panels can be designed for any organism with a known genome.
This webinar discusses the theory behind this technology and highlights the use of the rhAmpSeq system for identifying on- and off-target gene editing via CRISPR-Cas9.
Topics covered in this video:
- How does rhAmp PCR work?
- rhAmp PCR chemistry greatly reduces primer dimers and other non-specific amplification
- One system, many applications: Targeted GBS, CRISPR Genome Editing, Human Disease Research
- rhAmpSeq reagents
- Key benefit: Minimize primer-dimer formation
- Key benefit: Degenerate bases allowed
- Effects of mismatch in 1st PCR primer causing allelic drop out
- Repeat experiment with N base design
- Detecting on and off target gene editing events
- rhAmpSeq provides a simple high throughput workflow for targeted resequencing
- rhAmpSeq system for CRISPR: off-target NHEJ editing
- Validation of HiFi Cas9 System using rhAmpSeq system
- Off-target analysis: Alt-R HiFi Cas9 Nuclease
- Modified ssODNs improve HDR rates
- Advantages of rhAmpSeq HT protocol
Published on: September 20, 2019