Competitive PCR-DGGE analysis of bacterial mixtures: an internal standard and an appraisal of template enumeration accuracy.
Analysis of polymerase chain reaction (PCR) amplified 16S rDNA fragments from environmental samples by denaturing gradients of chemicals or heat [denaturing gradient gel electrophoresis (DGGE) and thermal gradient gel electrophoresis (TGGE)] within polyacrylamide gels is a popular tool in microbial ecology. Difficulties in acceptance of the technique and interpretation of the results remain, due to its qualitative nature. In this study we have addressed this problem by the construction and evaluation of a quantitative standard for incorporation into test DNA samples. The standard was based on a naturally occurring 16S rRNA gene carried by the X-endosymbiont of the psyllid Anomoneura mori, a gamma-proteobacterium. This sequence is the most AT-rich 16S rDNA gene recovered from any cultured organism or environmental sample described to date, and a specifically amplified rDNA fragment denatured under exceptionally low stringency denaturing conditions. The native sequence was modified to incorporate perfect matches to the PCR primers used. The efficiency of amplification of this standard in comparison to a range of 16S rDNA sequences and the errors involved in enumerating template molecules under a range of PCR conditions are demonstrated and quantified. Tests indicated that highly accurate counts of released target molecules from a range of bacterial cells could be achieved in both laboratory mixtures and compost.
Brüggemann
J
J
Center for Environmental Biotechnology, University of Tennessee, 10515 Research Dr., Suite 300, Knoxville, TN 37932-2575, USA.
Stephen
J R
JR
Chang
Y J
YJ
Macnaughton
S J
SJ
Kowalchuk
G A
GA
Kline
E
E
White
D C
DC
D000421:Alcaligenes / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D016969:Desulfovibrio vulgaris / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D020560:Proteobacteria / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012680:Sensitivity and Specificity; D020614:Shewanella putrefaciens / Q000235:genetics
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S