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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.

With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the "ideal biocatalyst" for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of "sequence space" that is not addressed by traditional screening.

Numerous gene-specific PCR methods have been developed for the cultivation-independent discovery of novel genes from complex environmental DNA samples. The recovery of full-length genes is, however, technically challenging. Here, we present an efficient and relatively simple approach that combines magnetic bead capture with subtractive hybridization for the rapid and direct recovery of full-length target ORFs. When compared with other PCR-based techniques, a higher degree of specificity is achieved through the use of larger gene fragments during hybridization followed by several high-stringency washes. Together with the recent advances in environmental nucleic acid extraction techniques, this approach should allow for the further exploration of the metagenomic sequence space.