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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.
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病原微生物生物安全國(guó)家重點(diǎn)實(shí)驗(yàn)室
病原微生物生物安全國(guó)家重點(diǎn)實(shí)驗(yàn)室是2004年經(jīng)國(guó)家科技部批準(zhǔn)建設(shè)的國(guó)家重點(diǎn)實(shí)驗(yàn)室，主管部門是中國(guó)人民解放軍總后勤部衛(wèi)生部，依托單位是軍事醫(yī)學(xué)科學(xué)院。實(shí)驗(yàn)室主任曹務(wù)春研究員，副主任楊瑞馥研究員、祝慶余研究員、曹誠(chéng)研究員。
實(shí)驗(yàn)室以病原微生物生物安全為研究方向，以國(guó)家生物安全相關(guān)的病原微生物為研究對(duì)象，重點(diǎn)開(kāi)展病原微生物的發(fā)現(xiàn)、預(yù)警、檢測(cè)和防御相關(guān)的理論和技術(shù)研究，主要圍繞病原微生物偵察、預(yù)警，病原微生物的快速檢驗(yàn)、鑒定，新傳染病的發(fā)現(xiàn)與追蹤，重要病原微生物致病機(jī)理與防治基礎(chǔ)等四個(gè)方面開(kāi)展研究。
實(shí)驗(yàn)室依托軍事醫(yī)學(xué)科學(xué)院有生物學(xué)、基礎(chǔ)醫(yī)學(xué)、公共衛(wèi)生與預(yù)防醫(yī)學(xué)3個(gè)國(guó)家博士后流動(dòng)站可招收博士后人員，有微生物學(xué)、病原生物學(xué)、免疫學(xué)、流行病與衛(wèi)生統(tǒng)計(jì)學(xué)、軍事預(yù)防醫(yī)學(xué)、遺傳學(xué)、生物信息學(xué)7個(gè)博士學(xué)位授權(quán)學(xué)科和10個(gè)碩士學(xué)位授權(quán)學(xué)科可招收博士、碩士研究生。設(shè)有細(xì)菌學(xué)、病毒學(xué)、立克次體學(xué)、微生物毒素學(xué)、分析微生物學(xué)和微生物基因組學(xué)等專業(yè)實(shí)驗(yàn)室，涵蓋了病原微生物的所有分枝學(xué)科。
實(shí)驗(yàn)室擁有5個(gè)國(guó)家級(jí)中心和專業(yè)實(shí)驗(yàn)室，包括國(guó)家生物危害防護(hù)裝備工程中心、國(guó)家生物醫(yī)學(xué)分析中心分析微生物實(shí)驗(yàn)室、國(guó)家生物醫(yī)學(xué)分析中心DNA合成與序列分析實(shí)驗(yàn)室、國(guó)家艾滋病確認(rèn)實(shí)驗(yàn)室、國(guó)家土拉、類鼻疽菌種保藏實(shí)驗(yàn)室；5個(gè)全軍研究中心和重點(diǎn)實(shí)驗(yàn)室，包括全軍微生物檢測(cè)研究中心、全軍艾滋病檢測(cè)中心、全軍疾病監(jiān)測(cè)中心、全軍分子遺傳學(xué)重點(diǎn)實(shí)驗(yàn)室、全軍媒介生物防治重點(diǎn)實(shí)驗(yàn)室。
實(shí)驗(yàn)室掛靠5個(gè)國(guó)家和3個(gè)全軍專業(yè)學(xué)會(huì)，包括中國(guó)微生物學(xué)會(huì)病毒學(xué)專業(yè)委員會(huì)、中國(guó)微生物學(xué)會(huì)分析微生物專業(yè)委員會(huì)、中國(guó)生物工程學(xué)會(huì)醫(yī)藥生物技術(shù)專業(yè)委員會(huì)、中國(guó)動(dòng)物學(xué)會(huì)寄生蟲專業(yè)委員會(huì)、中國(guó)昆蟲學(xué)會(huì)醫(yī)學(xué)昆蟲學(xué)專業(yè)委員會(huì)、全軍生物防護(hù)專業(yè)委員會(huì)、全軍生物技術(shù)專業(yè)委員會(huì)、全軍流行病學(xué)專業(yè)委員會(huì)等。編輯出版《生物技術(shù)通訊》、《中國(guó)消毒學(xué)雜志》、《寄生蟲與醫(yī)學(xué)昆蟲學(xué)報(bào)》等全國(guó)性雜志。
通過(guò)加強(qiáng)建設(shè)，力爭(zhēng)建設(shè)成為在病原微生物生物安全研究領(lǐng)域組織應(yīng)用基礎(chǔ)研究、聚集和培養(yǎng)優(yōu)秀科學(xué)家、開(kāi)展學(xué)術(shù)交流的重要基地，構(gòu)建一批國(guó)內(nèi)領(lǐng)先、國(guó)際一流的研究技術(shù)平臺(tái)，滿足承擔(dān)和完成國(guó)家重大科研任務(wù)的需要。
