NDLI: PCR-enriched cDNA pool method for cloning of gene homologues

Content Provider	Springer Nature Link
Author	Lang, Minglin Zhang, Yuxiu Guan, Ziqiu Chai, Tuanyao
Copyright Year	2005
Abstract	We have developed a PCR-enriched cDNA pooling method for enrichment of the complete 5′ ends of the target homology cDNA fragments with just 1 conserved region needed. By using reverse transcription and a few rounds of PCR amplification, a full-length cDNA population flanked by T7 and M13 primers was generated. Multiple complete 5′ ends of cDNA members of a gene family can subsequently be enriched via PCR with M13 and degenerate primer mix priming at the 5′ end and the conserved region, and they migrate as a single dense band when separated on an agarose gel. The enriched homologous cDNA fragments could be separated for subsequent cloning and sequencing. The main advantages of our method are its speediness, simplicity, and cost-effectiveness. The method has been successfully applied to the cloning of members of the cation-efflux family inBrassica juncea L. and the natural resistance-associated macrophage protein family inThlaspi caerulescens, which demonstrates that this novel approach permits rapid isolation of novel interspecific gene orthologues. It could also be easily adapted to highly specific cloning of gene homologues identified in target genomes.
Starting Page	219
Ending Page	226
Page Count	8
File Format	PDF
ISSN	07359640
Journal	Plant Molecular Biology Reporter
Volume Number	23
Issue Number	3
e-ISSN	15729818
Language	English
Publisher	Springer-Verlag
Publisher Date	2012-09-03
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	cation-efflux protein family cloning of gene orthologues gene family PCR-enriched cDNA pooling Plant Sciences Plant Breeding/Biotechnology Proteomics Metabolomics Bioinformatics
Content Type	Text
Resource Type	Article
Subject	Plant Science Molecular Biology

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