最近一期的PLoS ONE雜志（2007-11-14）刊登了我國軍事醫(yī)學(xué)科學(xué)院微生物流行病研究所病原微生物生物安全國家重點實驗室童貽剛研究員發(fā)明的多外顯子cDNA的直接快速克隆技術(shù)——“基因組DNA剪接”（genomic DNA splicing, GDS）技術(shù)（http://www.plosone.org/doi/pone.0001179）,。該技術(shù)克服了常規(guī)cDNA克隆方法的諸多弊端（RNA提取,，cDNA制備），直接從任何組織來源的基因組DNA中快速克隆任意全長的cDNA序列,。
由于生物信息學(xué)的高速發(fā)展,，人類以及各種模式生物的基因組全序列均已公開,，可以在網(wǎng)上自由獲取。然而對于生物醫(yī)學(xué)研究者來說,，要研究某個基因,，通常需要先將其克隆。由于哺乳動物基因組中存在大量的內(nèi)含子序列,，大多數(shù)的基因都相當(dāng)長,，動輒幾萬堿基對，對其進(jìn)行基因操作相當(dāng)麻煩（常規(guī)克隆載體對插入片段的大小有限制,，過大的片段不易克隆,，即使克隆成功也容易產(chǎn)生缺失；此外大片段含有過多的限制性酶切位點,，使后續(xù)的基因操作變得十分困難）,。
對該問題的解決辦法就是使用cDNA代替基因組DNA。由于完整的cDNA序列在自然界中并不存在,，需要采用人工的方法,，將細(xì)胞中的mRNA進(jìn)行逆轉(zhuǎn)錄才能獲得，因此克隆cDNA序列必須經(jīng)過mRNA的制備以及逆轉(zhuǎn)錄等步驟,，完成這些步驟均需使用特殊的試劑盒,，而且在我們的實驗環(huán)境中RNA酶無處不在（RNA酶為實驗室常用的試劑,，在所有的動物細(xì)胞中表達(dá)），而且十分頑固,，難以滅活,，很容易給cDNA的制備造成麻煩。常規(guī)cDNA克隆方法的另外一個缺點就是常常需要獲取特定的動物組織,，因為經(jīng)過分化的不同器官組織表達(dá)的基因不同,，許多基因僅在一些特殊的組織中表達(dá)，要順利的克隆這些基因,，選擇高表達(dá)的組織往往是必要的,，但是有些組織在動物機體中數(shù)量稀少，給克隆相應(yīng)的基因造成很大的問題,。
童貽剛等發(fā)明的genomic DNA splicing全長cDNA克隆技術(shù),，克服了上述所有的缺點。該技術(shù)無需采用逆轉(zhuǎn)錄的方法制備cDNA,，因此也就無需制備RNA,，更不用操作特定基因高表達(dá)的器官組織。整個實驗過程簡單快速,，僅使用常規(guī)的實驗室設(shè)備和試劑,，即可輕易完成多外顯子cDNA的全長序列的克隆。
原始出處:
Received: August 22, 2007; Accepted: October 18, 2007; Published: November 14, 2007
Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA
Xiaoping An1#, Jun Lu2#, Jian-dong Huang3*, Baozhong Zhang1, Dabin Liu1, Xin Zhang1, Jinhui Chen1, Yusen Zhou1, Yigang Tong1*
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.
Methodology
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.
Conclusions
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.
