Influence of primer mismatch and microdiversity on DGGE results: a case study with SAR11

Aquatic Microbial Ecology

Ramon Massana¹  Josep M. Gasol¹  Olga Sánchez¹  Carlos Pedrós-Alió¹  Jordi Mas¹  Vanessa Balagué¹ 

关键词: DGGE;    SAR11;    Microdiversity;    Primers;    Methodology;   

DOI  :  10.3354/ame01267

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: Although SAR11 is usually the dominant bacterial group in most marine ecosystems when analyzed with clone libraries and fluorescence in situ hybridization, it is often not retrieved in studies where denaturing gradient gel electrophoresis (DGGE) has been used. We analyzed the microdiversity of SAR11 in Blanes Bay (NW Mediterranean) and we suggest that the high evenness of multiple microdiverse phylotypes, none of which being particularly dominant, is the probable reason for this methodological discrepancy. We used seeding experiments in which different amounts of 2 SAR11-affiliated clones were mixed with DNA from an environmental sample obtained from the Blanes Bay Microbial Observatory. Two primer sets differing at 2 base positions produced DGGE images that varied in their SAR11 detection threshold concentration. Our results show that primer mismatches and/or the presence of faint bands due to microdiversity could explain why SAR11 is frequently not retrieved from DGGE gels.

【 授权许可】

Unknown