ArticleNucleic acids research2019
μLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome.
Article in Nucleic acids research, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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3 citing papers in PubMed, 8 citations in OpenAlex.
- Detection of EGFR mutations at pM concentration in ten minutes using a microfluidic concentration and separation module.Biomedical microdevices · 2025Article
- Hybridization-based DNA biosensing with a limit of detection of 4 fM in 30 s using an electrohydrodynamic concentration module fabricated by grayscale lithography.Biomicrofluidics · 2022Article
- Recent Advances in Strategies for the Cloning of Natural Product Biosynthetic Gene Clusters.Frontiers in bioengineering and biotechnology · 2021Review
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Authors and funding
14 authors at 3 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the μLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/μl for 50 kb fragments and an analytical time of 50 min. Then, μLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with μLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with μLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.
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