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Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA
1 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China, 2 Beijing YouAn Hospital, Capital Medical University, Beijing, China, 3 Department of Biochemistry, The University of Hong Kong, Hong Kong Special Administrative Region (SAR), China
Abstract
Background
Polymerase chain reaction (PCR) is extensively applied in gene cloning. But due to the existence of introns, low copy number of particular genes and high complexity of the eukaryotic genome, it is usually impossible to amplify and clone a gene as a full-length sequence directly from the genome by ordinary PCR based techniques. Cloning of cDNA instead of genomic DNA involves multiple steps: harvest of tissues that express the gene of interest, RNA isolation, cDNA synthesis (reverse transcription), and PCR amplification. To simplify the cloning procedures and avoid the problems caused by ubiquitously distributed durable RNases, we have developed a novel strategy allowing the cloning of any cDNA or open reading frame (ORF) with wild type sequence in any spliced form from a single genomic DNA preparation.
Our “Genomic DNA Splicing” technique contains the following steps: first, all exons of the gene are amplified from a genomic DNA preparation, using software-optimized, highly efficient primers residing in flanking introns. Next, the tissue-specific exon sequences are assembled into one full-length sequence by overlapping PCR with deliberately designed primers located at the splicing sites. Finally, software-optimized outmost primers are exploited for efficient amplification of the assembled full-length products.
The “Genomic DNA Splicing” protocol avoids RNA preparation and reverse transcription steps, and the entire assembly process can be finished within hours. Since genomic DNA is more stable than RNA, it may be a more practical cloning strategy for many genes, especially the ones that are very large and difficult to generate a full length cDNA using oligo-dT primed reverse transcription. With this technique, we successfully cloned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons.
附：
病原微生物生物安全国家重点实验室
病原微生物生物安全国家重点实验室是2004年经国家科技部批准建设的国家重点实验室，主管部门是中国人民解放军总后勤部卫生部，依托单位是军事医学科学院。实验室主任曹务春研究员，副主任杨瑞馥研究员、祝庆余研究员、曹诚研究员。
实验室以病原微生物生物安全为研究方向，以国家生物安全相关的病原微生物为研究对象，重点开展病原微生物的发现、预警、检测和防御相关的理论和技术研究，主要围绕病原微生物侦察、预警，病原微生物的快速检验、鉴定，新传染病的发现与追踪，重要病原微生物致病机理与防治基础等四个方面开展研究。
实验室依托军事医学科学院有生物学、基础医学、公共卫生与预防医学3个国家博士后流动站可招收博士后人员，有微生物学、病原生物学、免疫学、流行病与卫生统计学、军事预防医学、遗传学、生物信息学7个博士学位授权学科和10个硕士学位授权学科可招收博士、硕士研究生。设有细菌学、病毒学、立克次体学、微生物毒素学、分析微生物学和微生物基因组学等专业实验室，涵盖了病原微生物的所有分枝学科。
实验室拥有5个国家级中心和专业实验室，包括国家生物危害防护装备工程中心、国家生物医学分析中心分析微生物实验室、国家生物医学分析中心DNA合成与序列分析实验室、国家艾滋病确认实验室、国家土拉、类鼻疽菌种保藏实验室；5个全军研究中心和重点实验室，包括全军微生物检测研究中心、全军艾滋病检测中心、全军疾病监测中心、全军分子遗传学重点实验室、全军媒介生物防治重点实验室。
实验室挂靠5个国家和3个全军专业学会，包括中国微生物学会病毒学专业委员会、中国微生物学会分析微生物专业委员会、中国生物工程学会医药生物技术专业委员会、中国动物学会寄生虫专业委员会、中国昆虫学会医学昆虫学专业委员会、全军生物防护专业委员会、全军生物技术专业委员会、全军流行病学专业委员会等。编辑出版《生物技术通讯》、《中国消毒学杂志》、《寄生虫与医学昆虫学报》等全国性杂志。
通过加强建设，力争建设成为在病原微生物生物安全研究领域组织应用基础研究、聚集和培养优秀科学家、开展学术交流的重要基地，构建一批国内领先、国际一流的研究技术平台，满足承担和完成国家重大科研任务的需要。
