The Influence Of The Complexity Of Templates On The Pcr Bias

PCR technique has been a useful tool in the establishment of many analytical methods in microbial ecology and enhanced the development of microbial molecular ecology. Although it helped a lot in the community analysis, it can also introduce wrong information like PCR artifacts and bias which would interfere significantly in community analysis. Annealing temperature and cycle number are major elements that contributes to the bias of the amplification, besides, flanking area of the template also affect the efficiency of former cycles.The microbial model was made by combining same quantity amount of different kinds of bacteria(Pseudomonas putida KT2440, Escherichia coli DH5a, Bacillus subtilis168, Brevibacterium sp., Agrobacterium sp., Cellulosimicrobium sp., Microbacterium sp., Rhodococcus sp., Acetobacter pasteurianus sp. and Pseudomonas sp.), and mix them with autoclaved soil samples to serve as research target. The PCR bias was detected by ARDRA and the effect of simplification of template DNA on bias was analyzed. Template DNA was simplified either by partial digestion with Sau3A I or by ultrasonic treatment, we compared the effects of both treatments on PCR bias and applied ultrasonic treatment into microbiological community analysis of different manure treated red soil samples with DGGE and RISA analysis. In addition, the DNA recovery method based on nucleic acid hybridization was initially established.For group A in which total bacteria DNA was used directly as template, six strains were detected representing60%of the total ten species’ community; the detection rate is90%in group B with bacteria DNA partially predigested as template, uniformity of detected proportion strains is higher in group B, and the proportion of B. subtilis168and Rhodococcus sp. are close to the cell ratios in it. In group C, autoclaved soil samples was mixed with cell culture and total DNA as templates, bacteria detection rate is60%for it; the detection rate raised up to80%when the bacteria DNA in group C was predigested to produce group D, and the proportion of P. putida KT2440is close to its cell ratios in the group. The homogeneity in group B and D are better than those in group A and C, which indicates reduction in bias can be achieved by simplifying the templates. However, Pseudomonas sp. was not detected in any of the four groups, which might be caused by certain restriction of the method and thus further improvement is needed. Significant differences exist between both group A and C and group B and D, which might be caused by complicated chemical compounds naturally exists in soil like humic acid that affect totai DNA extraction and PCR reaction, and further affected the revealing of soil microbial composition.We mixed total DNA of four kinds of bacteria:Agrobacterium sp., Rhodococcus sp., Cellulosimicrobium sp. and Microbacterium sp. at the ratio of1:5:10:20as multi-template for bias study, the detection rate of Agrobacterium sp. is higher up to45.59%, far more than its DNA contents ratio2.78%. Enzymatic digestion and ultrasonic treatment can reduce the bias, but their effect do vary from each other, the Rhodococcus sp. with the DNA contents ratio of13.89%was detected as1.75%in enzymatic digestion and23.67%by ultrasonic treatment both rates are far from DNA contents ratio;55.56%is the DNA contents ratio of Microbacterium sp. in the complex, but it’s corresponding actual detection rate was grow up to70.18%in ultrasonic treatment and fall down to45.71%in enzymatic digestion group.DGGE analysis results of four different fertilization treated systems (CK:no fertilizer, N:N fertilizer, N+OM:N fertilizer+straw, OM:straw+pig manure) on red soil samples show no significant difference on band number and distribution among them; although variance in band pattern was observed between direct use of extracted total DNA template and ultrasonicating of total DNA, their differences were not significant when compared to previously treated strain model. Even if the ultrasonic processed total DNA fragments were no longer than4kb, they are far more complicated than short V3fragment, the complexity is not suitable for the primers to adhere to, and thus hard to be properly amplified out.RISA analysis using directly extracted DNA and ultrasonic treated DNA as templates, difference in band numbers and strength of the two groups. Community structure between CK and N, N+OM and OM are similar to each other respectively in directly extracted DNA groups. However, in the ultrasonic treated group the community structure of CK, N and N+OM are similar, which far apart with OM. The difference in RISA originated from different treatment indicated that template DNA structures do have important effects on RISA results, which further affect microbial community analysis. The method based on nucleic acid hybridization was initially established, which streptavidin-coated paramagnetic particles as the solid phase connected with biotin labled DNA probe. The targets DNA were recovery from the ultrasonic treated total DNA.