Abstract:
BACKGROUND:Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. RESULTS:We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. CONCLUSIONS:A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.
journal_name
BMC Biotechnoljournal_title
BMC biotechnologyauthors
Yadav RK,Barbi F,Ziller A,Luis P,Marmeisse R,Reddy MS,Fraissinet-Tachet Ldoi
10.1186/1472-6750-14-80subject
Has Abstractpub_date
2014-09-03 00:00:00pages
80issn
1472-6750pii
1472-6750-14-80journal_volume
14pub_type
杂志文章abstract:BACKGROUND:There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually con...
journal_title:BMC biotechnology
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journal_title:BMC biotechnology
pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
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