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Article: Rapid assembly of multiple-exon cDNA directly from genomic DNA

TitleRapid assembly of multiple-exon cDNA directly from genomic DNA
Authors
Issue Date2007
PublisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.action
Citation
Plos One, 2007, v. 2 n. 11 How to Cite?
AbstractBackgrouud. 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 genamic 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 doned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons. © 2007 An et al.
Persistent Identifierhttp://hdl.handle.net/10722/68102
ISSN
2015 Impact Factor: 3.057
2015 SCImago Journal Rankings: 1.395
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
National High Technology Research and Development Program of China2001AA215361
Funding Information:

This work is supported by National High Technology Research and Development Program of China (2001AA215361). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

 

DC FieldValueLanguage
dc.contributor.authorAn, Xen_HK
dc.contributor.authorLu, Jen_HK
dc.contributor.authorHuang, JDen_HK
dc.contributor.authorZhang, Ben_HK
dc.contributor.authorLiu, Den_HK
dc.contributor.authorChen, Jen_HK
dc.contributor.authorZhou, Yen_HK
dc.contributor.authorTong, Yen_HK
dc.date.accessioned2010-09-06T06:01:21Z-
dc.date.available2010-09-06T06:01:21Z-
dc.date.issued2007en_HK
dc.identifier.citationPlos One, 2007, v. 2 n. 11en_HK
dc.identifier.issn1932-6203en_HK
dc.identifier.urihttp://hdl.handle.net/10722/68102-
dc.description.abstractBackgrouud. 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 genamic 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 doned the full-length wild type coding sequence of human polymeric immunoglobulin receptor, which is 2295 bp in length and composed of 10 exons. © 2007 An et al.en_HK
dc.languageengen_HK
dc.publisherPublic Library of Science. The Journal's web site is located at http://www.plosone.org/home.actionen_HK
dc.relation.ispartofPLoS ONEen_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong Licenseen_HK
dc.subject.meshBase Sequence-
dc.subject.meshDNA Primers-
dc.subject.meshDNA, Complementary - genetics-
dc.subject.meshDNA-Directed DNA Polymerase - metabolism-
dc.subject.meshExons-
dc.titleRapid assembly of multiple-exon cDNA directly from genomic DNAen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1932-6203&volume=2&issue=11&spage=1179&epage=&date=2007&atitle=Rapid+assembly+of >multiple-exon+cDNA+directly+from+genomic+DNAen_HK
dc.identifier.emailHuang, JD:jdhuang@hkucc.hku.hken_HK
dc.identifier.authorityHuang, JD=rp00451en_HK
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1371/journal.pone.0001179en_HK
dc.identifier.pmid18000550en_HK
dc.identifier.pmcidPMC2048664-
dc.identifier.scopuseid_2-s2.0-43149124874en_HK
dc.identifier.hkuros146696en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-43149124874&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume2en_HK
dc.identifier.issue11en_HK
dc.identifier.isiWOS:000207459100021-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridAn, X=7102948557en_HK
dc.identifier.scopusauthoridLu, J=26643231600en_HK
dc.identifier.scopusauthoridHuang, JD=8108660600en_HK
dc.identifier.scopusauthoridZhang, B=35338495100en_HK
dc.identifier.scopusauthoridLiu, D=24173553700en_HK
dc.identifier.scopusauthoridChen, J=24172961700en_HK
dc.identifier.scopusauthoridZhou, Y=24173981700en_HK
dc.identifier.scopusauthoridTong, Y=7202614688en_HK
dc.identifier.citeulike2243050-

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