Optimization of ITS rDNA Amplification for Fungi from Black Soil in North China  

作　　者：YU Gaobo WU Fengzhi 

机构地区：[1]College of Horticulture, Northeast Agricultural University, Harbin 150030, China

出　　处：《Journal of Northeast Agricultural University(English Edition)》2011年第1期25-32,共8页东北农业大学学报（英文版）

基　　金：Supported by National Natural Science Foundation of China (30571264);Nation Key Technology Program (2006BAD07B03)

摘　　要：In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was used to evaluate five factors（template,Mg2＋,dNTP,Taq DNA polymerase,and primer） from four levels.Subsequently,the optimal annealing temperature,annealing time,extension time and cycle numbers were evaluated.The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer,30 ng soil DNA template,3.0 mmol·L-1 Mg2＋,0.2 mmol·L-1 dNTPs,0.1 μmol·L-1 each forward and reverse primer,and 2.0 U Taq enzyme in 50 μL reaction volume.The optimal thermal cycling protocol consisted of initial melting at 94℃ for 5 min,followed by 35 cycles at 94℃ for 30 s,56℃ for 30 s,72℃ for 90 s,and a final extension of 72℃ for 10 min.In order to optimize polymerase chain reaction（PCR） amplification of the internal transcribed spacer（ITS） rDNA region from fungi found in black soil in North China,an orthogonal experimental design [L16（45）] was used to evaluate five factors（template,Mg2＋,dNTP,Taq DNA polymerase,and primer） from four levels.Subsequently,the optimal annealing temperature,annealing time,extension time and cycle numbers were evaluated.The results showed that the optimized PCR solution for amplification of ITS region comprised 5 μL 10× buffer,30 ng soil DNA template,3.0 mmol·L-1 Mg2＋,0.2 mmol·L-1 dNTPs,0.1 μmol·L-1 each forward and reverse primer,and 2.0 U Taq enzyme in 50 μL reaction volume.The optimal thermal cycling protocol consisted of initial melting at 94℃ for 5 min,followed by 35 cycles at 94℃ for 30 s,56℃ for 30 s,72℃ for 90 s,and a final extension of 72℃ for 10 min.

关 键 词：OPTIMIZATION PCR soil fungi ITS rDNA 

分 类 号：Q503[生物学—生物化学]