Study On The Factors Affecting Bias In Multitemplate PCR And Optimization Of The Amplification Conditions

The detection,identification,and characterization of microbial populations and their activities in environments are a great challenge to microbiologists.In the last decade,the use of 16S rRNAs or ribosomal DNAs(rDNAs) as molecular markers has become routine for microbial ecologists.Several different rRNA-based approaches have been used to characterize microbial communities,such as restriction fragment length polymorphism analysis(RFLP),denaturing gradient gel electrophoresis(DGGE),and temperature gradient gel electrophoresis(TGGE).Nearly every study applying these approaches reveals the existence of many novel microbial groups that can not be isolated by traditional culture-dependent techniques.Use of these methods with multitemplate PCR,however,can cause bias and artifacts that lead to overestimation of community diversity.In this paper we investigated the bias in multitemplate PCR with an eight-species model(Pseudomonas putida KT2440,Escherichia coli DH5α,Bacillus subtilis 168, Brevibacterium sp.,Agrobacterium sp.,Cellulosimicrobium sp.,Microbacterium sp.,and Rhodococcus sp.),the effects of DNA polymerases,PCR cycles,primers,annealing temperature,16S rDNA GC contents and DNA structure on PCR bias were investigated,then the optimization of conditions were used to analyze the diversity of eight-species model.Eight strains were mixed equally and the DNAs were extracted for PCR amplification to construct a 16S rDNA library.138 clones were analyzed by RFLP and a strong bias in multitemplate PCR was observed.P.putida KT2440 was preferentially amplified,the percentage of P.putida KT2440,Brevibacterium sp.,E.coli DH5α,B.subtilis 168 were 47.1%,34.8%,5.8%,1.4%respectively,whereas the other four strains can hardly be detected.Four strains were mixed equally to study the PCR bias,the result indicated Agrobacterium sp.was selectively amplified,it accounted for 94.8%,Rhodococcus sp. accounted for 2.6%,whereas Cellulosimicrobium sp.and Microbacterium sp.still could not be detected.To test to what extent can all strains be detected,Agrobacterium sp.,Rhodococcus sp., Cellulosimicrobium sp.and Microbacterium sp.were mixed at the ratios of 1:10:50:100 and subjected to PCR amplification.The results suggested all strains were detectable, Agrobacterium sp.accounted for 40.9%of the total products though its template comprised the least proportion.Thus,analysis based on multitemplate PCR could bring serious bias.Effects of PCR cycle numbers,DNA polymerases,PCR primer,PCR annealing temperature,DNA extraction efficiency,16S rDNA GC contents and DNA structure on multitemplate PCR bias were determined.The results suggested that PCR cycle number had slight effects on bias;DNA polymerases,PCR primer and 16S rDNA GC contents were not the main cause of bias,whereas the bias caused by primer mismatched was strongly dependent on the PCR annealing temperature;there were differences in the DNA extraction efficiency,but it was not the main cause of bias;DNA structure had considerable effects on bias,when the DNA were digested with Bsa29Ⅰor HindⅢ+XbaⅠ,the bias was reduced but the overamplification species changed,whereas the bias was reduced greatly and four strains were amplified equally when digested with Sau3AⅠ.Optimized conditions were used for ampilification of the eight-species model.DNA were digested with Sau3AⅠand 2-4Kb fragments were extracted and used as template, annealing at 45℃,running 20 cycles.The result showed that after condition optimization, PCR bias was still observed,but greatly decreased,it will lay the foundation for later research of conditions under which multitemplate PCR bias is minimized.