近年来应大规模基因组学和遗传学研究发展的需要,能够提供“高通量、低成本、低劳动量”测序服务的新一代大规模并行测序技术应运而生.辅助其应用的生物信息分析软件和实验方法也层出不穷.而在关联研究如癌症研究中有广泛应用的基于PCR的基因组学和遗传学研究方法,却因其研究的目标区域较短,而较少地受益于采用“鸟枪法测序”策略的新一代测序技术.尽管已提出诸如“芯片捕获法”、“目标选择法”等技术克服这一难题,但这些方法对仪器和实验室操作的额外要求限制了它们的应用和推广.本实验提出一种能简便克服这一难题的“边扩增边连接方法”(Ligation by Amplification,LBA):使用一对“公用接头”和两步PCR反应将多个目标区域随机连接起来,其随机连接而成的长链产物可被简易地随机片段化并在新一代测序仪上测序.使用专门设计的、包含公用接头的LBA引物,将人类基因组保守编码序列中两个癌症相关基因:BRCA1和BRCA2的外显子进行扩增,并将所得到的70个扩增产物在一步PCR过程中进行边扩增边连接.所得的长链LBA产物经随机片段化后制备成可被传统Sanger测序法和新一代边合成边测序技术测序的DNA文库,分别在ABI3730xl测序仪和Illumina/Solexa Genome Analyzer测序仪上进行测序.对目标序列的覆盖度、覆盖深度和单核苷酸多态性检测的有效性的生物信息学分析证明:运用本方法可高效地在新一代测序仪上进行基于PCR的重测序和遗传多态性研究,推动各种基于PCR的基因组学和遗传学研究的发展. In recent years, a large-scale massively parallel sequencing technology capable of providing “high-throughput, low-cost, low labor-saving” sequencing services has emerged in response to the needs of large-scale genomics and genetics research and development. Information analysis software and experimental methods are also emerging.While PCR-based genomics and genetics research methods are widely used in related research such as cancer research, but because of its shorter target area, and less benefit from the use of A Next-Generation Sequencing Technique for the “Shotgun Sequencing” Strategy Although techniques such as “chip capture” and “target selection” have been proposed to overcome this challenge, these methods add extra to instrumentation and laboratory operations Which limits their application and promotion.This experiment proposed a “Ligation by Amplification (LBA)” method that could easily overcome this problem: using a pair of “common joints” and two The step PCR reaction randomly links multiple target regions, and their randomly ligated long-chain products can be simply randomly fragmented and sequenced on a next generation sequencer using a specially designed LBA containing a common adapter Which amplifies the exons of two cancer-related genes BRCA1 and BRCA2 in the conserved coding sequence of the human genome and amplifies the resulting 70 amplicons while amplifying them in a one-step PCR. The long-chain LBA product was randomly fragmented to prepare a DNA library which can be sequenced by traditional Sanger sequencing and a new generation of sequencing-by-synthesis side sequencing technology, respectively, on the ABI3730xl sequencer and the Illumina / Solexa Genome Analyzer sequencer. Bioinformatics analysis of the coverage, coverage depth and the validity of single nucleotide polymorphism (SNP) detection demonstrated that PCR-based resequencing and genetic polymorphism research can be efficiently performed on a new generation of sequencer using this method, Promote the development of various PCR-based genomics and genetics research.