Evaluation of PCR amplification bias by terminal restriction fragment length polymorphism analysis of small-subunit rRNA and mcrA genes by using defined template mixtures of methanogenic pure cultures and soil DNA extracts

被引:244
作者
Lueders, T [1 ]
Friedrich, MW [1 ]
机构
[1] Max Planck Inst Terr Mikrobiol, D-35043 Marburg, Germany
关键词
D O I
10.1128/AEM.69.1.320-326.2003
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Terminal restriction fragment length polymorphism (T-RFLP) analysis is a widely used method for profiling microbial community structure in different habitats by targeting small-subunit (SSU) rRNA and also functional marker genes. It is not known, however, whether relative gene frequencies of individual community members are adequately represented in post-PCR amplicon frequencies as shown by T-RFLP. In this study, precisely defined artificial template mixtures containing genomic DNA of four different methanogens in various ratios were prepared for subsequent T-RFLP analysis. PCR amplicons were generated from defined mixtures targeting not only the SSU rRNA but also the methyl-coenzyme M reductase (mcrA/mrtA) genes of methanogens. Relative amplicon frequencies of microorganisms were quantified by comparing fluorescence intensities of characteristic terminal restriction fragments. SSU ribosomal DNA (rDNA) template ratios in defined template mixtures of the four-membered community were recovered absolutely by PCR-T-RFLP analysis, which demonstrates that the T-RFLP analysis evaluated can give a quantitative view of the template pool. SSU rDNA-targeted T-RFLP analysis of a natural community was found to be highly reproducible, independent of PCR annealing temperature, and unaffected by increasing PCR cycle numbers. Ratios of mcrA-targeted T-RFLP analysis were biased, most likely by PCR selection due to the degeneracy of the primers used. Consequently, for microbial community analyses, each primer system used should be evaluated carefully for possible PCR bias. In fact, such bias can be detected by using T-RFLP analysis as a tool for the precise quantification of the PCR product pool.
引用
收藏
页码:320 / 326
页数:7
相关论文
共 51 条
[1]  
AVANISSAGHAJANI E, 1994, BIOTECHNIQUES, V17, P144
[2]  
Berthelet M, 1996, FEMS MICROBIOL LETT, V138, P17
[3]   Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in pacific northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes [J].
Braker, G ;
Ayala-del-Río, HL ;
Devol, AH ;
Fesefeldt, A ;
Tiedje, JM .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2001, 67 (04) :1893-1901
[5]  
Brunk CF, 1998, APPL ENVIRON MICROB, V64, P5064
[6]   Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii [J].
Bult, CJ ;
White, O ;
Olsen, GJ ;
Zhou, LX ;
Fleischmann, RD ;
Sutton, GG ;
Blake, JA ;
FitzGerald, LM ;
Clayton, RA ;
Gocayne, JD ;
Kerlavage, AR ;
Dougherty, BA ;
Tomb, JF ;
Adams, MD ;
Reich, CI ;
Overbeek, R ;
Kirkness, EF ;
Weinstock, KG ;
Merrick, JM ;
Glodek, A ;
Scott, JL ;
Geoghagen, NSM ;
Weidman, JF ;
Fuhrmann, JL ;
Nguyen, D ;
Utterback, TR ;
Kelley, JM ;
Peterson, JD ;
Sadow, PW ;
Hanna, MC ;
Cotton, MD ;
Roberts, KM ;
Hurst, MA ;
Kaine, BP ;
Borodovsky, M ;
Klenk, HP ;
Fraser, CM ;
Smith, HO ;
Woese, CR ;
Venter, JC .
SCIENCE, 1996, 273 (5278) :1058-1073
[7]   Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern [J].
Casamayor, EO ;
Massana, R ;
Benlloch, S ;
Ovreås, L ;
Díez, B ;
Goddard, VJ ;
Gasol, JM ;
Joint, I ;
Rodríguez-Valera, F ;
Pedrós-Alió, C .
ENVIRONMENTAL MICROBIOLOGY, 2002, 4 (06) :338-348
[8]  
Chin KJ, 1999, APPL ENVIRON MICROB, V65, P2341
[9]   Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities [J].
Clement, BG ;
Kehl, LE ;
DeBord, KL ;
Kitts, CL .
JOURNAL OF MICROBIOLOGICAL METHODS, 1998, 31 (03) :135-142
[10]   Isolating PCR-quality DNA from human feces with a soil DNA kit [J].
Clement, BG ;
Kitts, CL .
BIOTECHNIQUES, 2000, 28 (04) :640-+