关键词: RNA捕获法, 高通量测序, 靶序列捕获

Abstract:

Objective We took epidermal growth factor receptor(egfr) gene promotor DNA as target fragment and established RNA capture assay which is very efficient for targeted genomic DNA enrichment and can be used in deep sequencing of target regions with second-generation sequencing.
Methods HeLa S3 cervical cancer cells were used as a model in the study. The genomes were extracted, digested with Mbo Ⅰ, filled ends, added with PE adapters, then 400 bp~1 000 bp size fragments were selected as DNA library. egfr promotor RNA was used to hybridize with the library. Hybridized fragments were selected by magnetic beads, PCR amplified for 15 cycles, and sequenced with Solexa second generation sequencing system.
Results The sequenced tags were analyzed by bioinformatics. There were 1 889 of 106 reads mapping to egfr gene promotor and only 2, 3, 3 reads mapping to each notch 1, platelet derived growth factor-B(pdgf-B) and src gene loci. We selected 106 the same long reads randomly from human genome as control group. The results showed that egfr fragments were enriched to 630 times by the RNA capture assay.
Conclusion RNA capture of target genome sequence is an efficient assay for high-throughput re-sequencing of targeted regions of genome. RNA capture and high-throughput sequencing technology can be applied to re-sequencing of target regions, including regions related to alternative splicing, nucleosome distribution, single nucleotide polymorphisms(SNPs) and loci of complex disease-related genes.

Key words: RNA capture, highthroughput sequencing, target capturing