rapid assembly of multiple-exon cdna directly from genomic dnamultiple-exon cdna直接从基因组dna的快速组装.pdfVIP

Rapid Assembly of Multiple-Exon cDNA Directly from Genomic DNA 1. 2. 3 1 1 1 1 1 1 Xiaoping An , Jun Lu , Jian-dong Huang *, Baozhong Zhang , Dabin Liu , Xin Zhang , Jinhui Chen , Yusen Zhou , Yigang Tong * 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 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 splic