Retrieval of full-length functional genes using subtractive hybridization magnetic bead capture.
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.
Meiring
Tracy
T
Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Cape Town, South Africa.
Mulako
Inonge
I
Tuffin
Marla I
MI
Meyer
Quinton
Q
Cowan
Donald A
DA
D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods; D054892:Metagenome; D056186:Metagenomics / Q000295:instrumentation / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D016366:Open Reading Frames / Q000235:genetics; D012680:Sensitivity and Specificity
D004247:DNA