最近一期的PLoS ONE杂志（2007-11-14）刊登了我国军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室童贻刚研究员发明的多外显子cDNA的直接快速克隆技术——“基因组DNA剪接”（genomic DNA splicing, GDS）技术（http://www.plosone.org/doi/pone.0001179）。该技术克服了常规cDNA克隆方法的诸多弊端（RNA提取，cDNA制备），直接从任何组织来源的基因组DNA中快速克隆任意全长的cDNA序列。
由于生物信息学的高速发展，人类以及各种模式生物的基因组全序列均已公开，可以在网上自由获取。然而对于生物医学研究者来说，要研究某个基因，通常需要先将其克隆。由于哺乳动物基因组中存在大量的内含子序列，大多数的基因都相当长，动辄几万碱基对，对其进行基因操作相当麻烦（常规克隆载体对插入片段的大小有限制，过大的片段不易克隆，即使克隆成功也容易产生缺失；此外大片段含有过多的限制性酶切位点，使后续的基因操作变得十分困难）。
对该问题的解决办法就是使用cDNA代替基因组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，将细胞中的mRNA进行逆转录才能获得，因此克隆cDNA序列必须经过mRNA的制备以及逆转录等步骤，完成这些步骤均需使用特殊的试剂盒，而且在我们的实验环境中RNA酶无处不在（RNA酶为实验室常用的试剂，在所有的动物细胞中表达），而且十分顽固，难以灭活，很容易给cDNA的制备造成麻烦。常规cDNA克隆方法的另外一个缺点就是常常需要获取特定的动物组织，因为经过分化的不同器官组织表达的基因不同，许多基因仅在一些特殊的组织中表达，要顺利的克隆这些基因，选择高表达的组织往往是必要的，但是有些组织在动物机体中数量稀少，给克隆相应的基因造成很大的问题。
童贻刚等发明的genomic DNA splicing全长cDNA克隆技术，克服了上述所有的缺点。该技术无需采用逆转录的方法制备cDNA，因此也就无需制备RNA，更不用操作特定基因高表达的器官组织。整个实验过程简单快速，仅使用常规的实验室设备和试剂，即可轻易完成多外显子cDNA的全长序列的克隆。
原始出处:
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.
