Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

• DOI: 10.1128/aem.04150-13
• Publication Date: 2014-01-18T03:29:04Z
• Authors: Yuki Morono; Takeshi Terada; Tatsuhiko Hoshino; Fumio Inagaki
• Keywords: Pyrosequencing

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption DNA extraction. With a commonly used extraction kit, roughly two-thirds of subseafloor sediment cells remain intact on average (i.e., the are not disrupted), indicating that analyses may be biased at step, prior to subsequent molecular analyses. To address this issue, we standardized new method using alkaline treatment heating. Upon with 1 M NaOH 98°C 20 min, over 98% in samples collected different depths were disrupted. However, integrity tests showed such strong heat also cleaved molecules into short fragments could amplified by PCR. Subsequently, optimized temperature conditions minimize fragmentation retain high efficiency. The best produced rate 50 80% from various retained sufficient amplification complete 16S rRNA gene ∼1,500 bp). yielded higher concentrations all tested compared extractions conventional kit-based approach. Comparative real-time PCR pyrosequencing bacterial archaeal genes an increase its diversity, suggesting it provides better analytical coverage communities than methods.