利用RNA捕获法在全基因组进行egfr启动子片段的富集

Enrichment of egfr Gene Promotor in Human Genome Wide by RNA Capture

目的以表皮生长因子受体(epidermal growth factor receptor,egfr)启动子片段为目标靶序列,用RNA捕获法在全基因组进行靶序列的富集,结合第二代测序技术,实现候选基因区段的深度测序。方法本研究以宫颈癌细胞系HeLa S3 egfr启动子为模型,提取基因组后用MboⅠ酶切,补平加PE接头,回收400 bp～1000 bp的DNA片段作为模板,并用egfr启动子RNA与制备的基因组模板杂交将egfr启动子片段富集,富集的DNA片段PCR扩增15轮后用Solexa高通量测序。结果通过生物信息学技术分析,从实验组中随机取总条数为106 reads,比对到egfr启动子片段reads条数为1 889条,而比对到随机挑选的notch 1、pdgf-B、src基因reads条数分别为2、3、3条。从人类全基因组随机取总条数为106的等长reads作为对照组,比对到notch 1、pdgf-B、src、egfr基因reads条数分别为3,2,3,2条。在本实验中egfr启动子片段通过RNA捕获法被富集了630倍。结论用本研究建立的RNA捕获法进行基因组靶序列捕获、富集、测序文库建立的技术路线可行,所建DNA文库可直接用于Solexa测序仪的测序。RNA捕获技术与第二代高通量测序技术结合能应用于靶区域的重测序,研究可变剪切、核小体分布,发现和分析新的单核苷酸多态性(singlenucleotide polymorphisms,SNPs)位点,寻找许多复杂疾病相关基因及位点,针对性强,简便易行,节约成本。

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.