Bacterial and Archaeal DNA Extracted from Inoculated Experiments: Implication for the Optimization of DNA Extraction from Deep-Sea Basalts

Abstract

Difficulties in efficient DNA extraction from deep-sea volcanic basalt, due to high metal concentration, complex organic matter, or sometimes the low biomass, have hampered the understanding of the significant biosphere both at and below the sea floor. In order to optimize the DNA extraction from basaltic rocks, sterilized basalts with different particle sizes and chemically synthesized goethite were inoculated with an iron oxidizer Marinobacter aquaeolei and an extreme halophilic archaeon Halobaculum gomorrense respectively, and extracted with several methods. Large variations in DNA yields by different extracting methods including FastDNA® spin for soil kit, GeneClean® for ancient DNA kit, UltraClean™ and traditional phenol-chloroform methods. Among the commercially available kits tested here, FAST spin kit and GeneClean® for ancient DNA kit yield 10 times more DNA than the UltraClean™ kit used. In combination with FAST spin kit, skim milk greatly enhanced the archaeal DNA yields. DNA extracting efficiency was low with the cell number lower than 1 × 10⁶ cells, but reached as high as 88% with a cell number of 1 × 10⁸ cells. On these points, different strategies should be taken into consideration for the DNA extraction from basalts, depending on original biomass and cell types anticipated. FAST spin kit could provide high quality bacterial DNA for downstream PCR whilst the combination of FAST spin kit with skim milk would greatly enhance the archaeal DNA yields. GeneClean® for ancient DNA kit is also recommended for archaeal DNA extraction from deep sea basalt due to its high yield.

Keywords:

• deep-sea basalt
• DNA extraction
• goethite
• skim milk

We thank Dr. E. Webb for providing the strain of M. aquaeolei VT8^T. And we thank B. Orcutt, G. Horn and B. Wilson for the helpful discussion and comments during the research. This work is supported by NSFC project (40602081) to Hongmei Wang, the 111 Project (B08030) to China University of Geosciences (Wuhan), the NSF Microbial Observatories program (MCB- 0348425) and the RIDGE 2000 program (OCE-0241791) grants to K.J.E.