Direct and efficient cloning of full-length genes from environmental DNA by RT-qPCR and modified TAIL-PCR

Abstract

Environmental DNA (eDNA) is defined as the total DNA that can be isolated from environmental samples. In total, therefore, eDNA includes a vast functional genes, and various approaches have been developed to retrieve full-length functional genes from eDNA. The efficiency of PCR amplification of eDNA is limited, however, because in truth, the net content of actual target functional genes is rather low in eDNA. To address this technical challenge, we developed a fast and effective approach to cloning full-length functional genes from eDNA. Two important modifications were made to existing PCR-based methods: first, a real-time quantitative PCR step was added to assess the difficulty of obtaining full-length genes; second, we improved the thermal asymmetric interlaced PCR program to make it more effective for cloning the flanking regions of known fragments that are present at low abundance in eDNA. Using this approach, five novel full-length functional genes with very low identity to known genes were cloned from environmental samples. This approach has great potential for allowing discovery of functional genes from environmental sources and may be broadly applicable to molecular biology research.