This article is part of the supplement: Validation methods for functional genome annotation

Open Access Research

Filtering "genic" open reading frames from genomic DNA samples for advanced annotation

Sara D'Angelo1, Nileena Velappan1, Flavio Mignone2, Claudio Santoro3, Daniele Sblattero3, Csaba Kiss1 and Andrew RM Bradbury1*

  • * Corresponding author: Andrew RM Bradbury amb@lanl.gov

  • † Equal contributors

Author affiliations

1 Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA

2 Department of Structural Chemistry and Inorganic Stereochemistry, School of Pharmacy, University of Milan, Milan, Italy

3 Department of Medical Sciences and IRCAD, University of Eastern Piedmont, Novara, Italy

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Citation and License

BMC Genomics 2011, 12(Suppl 1):S5  doi:10.1186/1471-2164-12-S1-S5

Published: 15 June 2011

Abstract

Background

In order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA) for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the “domainome”.

Results

In this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP), normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the “domainone” that is accessible.

Conclusions

The availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of functional genic ORFs. ORF libraries represent, moreover, a useful tool to proceed towards high-throughput functional annotation of newly sequenced genomes.