Subtractive hybridization magnetic bead capture: A new technique for the recovery of full‐length ORFs from the metagenome

Abstract

A number of PCR-based (i.e., sequence dependent) techniques are available for the recovery of full-length functional genes from genomic DNA preparations. These include semi-nested PCR, TAIL PCR and inverse PCR [5,6]. The implementation of these techniques may be technically challenging, particularly for metagenomic samples [7]. To expedite and simplify gene recovery, we have adapted magnetic bead capture and subtractive hybridization techniques [8] for the specific recovery of fulllength ORFs. The combination of these techniques paves the way for more effective recovery of full-length functional genes from metagenomic DNA samples.In this study we sought to develop a more direct and rapid gene recovery technique to avoid some of the problems associated with gene-specific PCR of metagenomic samples. The use of gene-specific labeled magnetic beads in a subtractive hybridization assay has been adapted from Jacobsen [8], who demonstrated the microscale detection of Pseudomonas fluorescens genomic DNA in soil using a lux gene fragment as the immobilized probe.We modified this technique to allow the specific recovery of a target gene rather than the entire genome. Broadly, multiple target genes are PCR-amplified from metagenomic DNA using gene-specific degenerate primers. The partial gene products of this amplification process (the 'driver DNA'; Box 1) are then immobilized to streptavidin-covered magnetic beads and used as hybridization probes to recover 'full-length' genes from metagenomic DNA samples (Fig. 1).The first step in the DNA preparation is the introduction of adapter priming sites. Adapter sequences were reconstructed and ligated to the 3´-A-tailed metagenomic DNA fragments ('tester DNA': Box 1). To ensure efficient adapter ligation, a directional cloning strategy was employed. The adapters were designed to be partially single stranded at the 5´ end, forcing the incorporation of the adapter priming sites in the correct orientation. Prior to hybridization, the priming sites must be reconstructed by blunt-ending the adaptor 5´ overhang.There is potentially no limitation to the nature of the environmental samples that may be used. However, consideration must be given to the chemical and biochemical properties of the sample, and the way in which these It is now widely accepted that classical microbiological methods provide only limited access to the true microbial biodiversity (less than 1%) [1]. The desire to access a higher proportion of the metagenome [2] has led to the development of efficient environmental nucleic acid extraction technologies and to a range of sequence-dependent and sequence-independent gene discovery techniques [3]. These methods [2, 4] avoid many of the limitations of culture-dependent gene targeting.

The application of magnetic bead hybridization for the recovery and STR amplification of degraded and inhibited forensic DNA

“…Hybridization procedures work best for degraded DNA due to the better efficiency of capture of DNA fragments and improved amplification [21]. The quantity of beads used is crucial in controlling sensitivity of the hybridization reaction and the quality of the isolated DNA.…”

Mining the Metabiome: Identifying Novel Natural Products from Microbial Communities

“…Increasing the biosynthetic density of metagenomic libraries will therefore translate directly into more efficient screening. The majority of existing enrichment techniques rely on some variation of probe hybridization (Kalyuzhnaya et al, 2006; Meyer et al, 2007; Morimoto and Fujii, 2009; Uchiyama et al, 2005; Zhang et al, 2009). Recently, enrichment strategies based on genetic complementation have shown promise as tools for library scale enrichments.…”

Metagenomics in animal gastrointestinal ecosystem: a microbiological and biotechnological perspective

“…Growing methane-oxidizing anaerobic microbial communities on 15 N-labelled ammonium as N-source has also been shown to be a sensitive and fast way to detect growth of extremely slow-growing microorganisms in different sediment systems [77]. However, information is not currently available on applications of these techniques for enrichment of slow-growing rumen or GI bacteria.…”

“…An ideal procedure for recovering nucleic acids from environmental samples should meet several criteria like, high and unbiased recovery of nucleic acids representative of the entire microbial community, large enough size of pure nucleic acids (RNA/DNA) sequences, and simplicity of the extraction protocols [14]. However, owing to the physiochemical diversity in matrices serving as microbial habitats, there is not any universally applicable protocol for isolating the microbial nucleic acids and therefore, different extraction protocols have been in use [15,16]. The principal strategies for the recovery of metagenomic DNA include the microbial recovery and their cell lysis [17].…”