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A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations


With rapid advances in sequencing technologies, tens of millions of DNA variants have now been discovered in the human population. However, there are currently no experimental methods available for examining the impact of DNA variants in a high-throughput fashion. As a result, we have no functional data on the vast majority of these variants, which is a major roadblock to generating novel biological insights and developing new disease prevention therapeutic strategies. To address this issue, we have successfully developed the first massively-parallel site-directed mutagenesis approach, Clone-seq, to leverage the power of next-generation sequencing to generate a large number of mutant alleles in a fast and cost-effective manner. In conjunction with Clone-seq, we established a high-throughput comparative interactome-scanning pipeline to experimentally elucidate the effect of variants on protein stability and interactions. Additionally, Clone-seq can be used to generate clones for all DNA variants, including those in non-coding regions.


Vyšlo v časopise: A Massively Parallel Pipeline to Clone DNA Variants and Examine Molecular Phenotypes of Human Disease Mutations. PLoS Genet 10(12): e32767. doi:10.1371/journal.pgen.1004819
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004819

Souhrn

With rapid advances in sequencing technologies, tens of millions of DNA variants have now been discovered in the human population. However, there are currently no experimental methods available for examining the impact of DNA variants in a high-throughput fashion. As a result, we have no functional data on the vast majority of these variants, which is a major roadblock to generating novel biological insights and developing new disease prevention therapeutic strategies. To address this issue, we have successfully developed the first massively-parallel site-directed mutagenesis approach, Clone-seq, to leverage the power of next-generation sequencing to generate a large number of mutant alleles in a fast and cost-effective manner. In conjunction with Clone-seq, we established a high-throughput comparative interactome-scanning pipeline to experimentally elucidate the effect of variants on protein stability and interactions. Additionally, Clone-seq can be used to generate clones for all DNA variants, including those in non-coding regions.


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Štítky
Genetika Reprodukčná medicína

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PLOS Genetics


2014 Číslo 12
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