Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance

Yong Feng Wang, Fang Qiu Zhang, Ji Dong Gu*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Denaturing gradient gel electrophoresis (DGGE) is a powerful technique to reveal the community structures and composition of microorganisms in complex natural environments and samples. However, positive and reproducible polymerase chain reaction (PCR) products, which are difficult to acquire for some specific samples due to low abundance of the target microorganisms, significantly impair the effective applications of DGGE. Thus, nested PCR is often introduced to generate positive PCR products from the complex samples, but one problem is also introduced: The total number of thermocycling in nested PCR is usually unacceptably high, which results in skewed community structures by generation of random or mismatched PCR products on the DGGE gel, and this was demonstrated in this study. Furthermore, nested PCR could not resolve the uneven representative issue with PCR products of complex samples with unequal richness of microbial population. In order to solve the two problems in nested PCR, the general protocol was modified and improved in this study. Firstly, a general PCR procedure was used to amplify the target genes with the PCR primers without any guanine cytosine (GC) clamp, and then, the resultant PCR products were purified and diluted to 0.01 μg ml-1. Subsequently, the diluted PCR products were utilized as templates to amplify again with the same PCR primers with the GC clamp for 17 cycles, and the products were finally subjected to DGGE analysis. We demonstrated that this is a much more reliable approach to obtain a high quality DGGE profile with high reproducibility. Thus, we recommend the adoption of this improved protocol in analyzing microorganisms of low abundance in complex samples when applying the DGGE fingerprinting technique to avoid biased results.

Original languageEnglish
Pages (from-to)5655-5663
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume98
Issue number12
DOIs
StatePublished - Jun 2014
Externally publishedYes

Keywords

  • Anammox bacteria
  • Complex samples
  • DGGE
  • Improvement
  • Low abundance population
  • Protocol

Fingerprint Dive into the research topics of 'Improvement of DGGE analysis by modifications of PCR protocols for analysis of microbial community members with low abundance'. Together they form a unique fingerprint.

Cite this