光皮桦EST-SSR PCR反应体系的优化

为获得稳定的聚合酶链式反应（PCR）产物，采用正交设计L25（56）对光皮桦Betula luminifera表达序列标签-简单重复序列聚合酶链式反应（EST-SSR PCR）反应体系中的5个因素:DNA模板浓度，引物浓度，镁离子（Mg2+）浓度，三磷酸碱基脱氧核苷酸（dNTPs）浓度和DNA聚合酶量进行优化，每个因素设计5个水平。优化试验结果表明:光皮桦EST-SSR最佳PCR反应体系:4.0 mgL－1DNA模板，0.500 molL－1引物，1.250 mmolL－1 Mg2+，0.250 mmolL－1 dNTPs，0.072 5 16.67 mkatL－1 rTaq酶。通过梯度PCR试验筛选得到相应引物的最佳退火温度。对优化出的EST-SSR PCR反应体系进行稳定性检测，结果均能获得稳定、清晰可辨的DNA谱带。图6表3参15

Optimization of EST-SSR PCR reaction system for Betula luminifera

To obtain stable PCR product，providing the basis for further study，orthogonal design was used to optimize an Expressed Sequence Tags （EST）-Simple Sequence Repeat （SSR） Polymerase Chain Reaction （PCR） amplification system for Betula luminifera in terms of five factors containing rTaq DNA polymerase，Mg2+，DNA template，dNTPs and primers in five levels. Results showed an optimal EST-SSR PCR system for B. luminifera was 4 mgL-1 DNA template，0.500 molL-1 primers，1.250 mmolL-1 Mg2+，0.250 mmolL-1 dNTPs，and 0.072 516.67 mkatL-1 rTaq DNA polymerase. And the optimal annealing temperature to each primers for SSR PCR reaction system is determined by gradient PCR. Using the optimal reaction system，stable and clear polymorphic bands were acquired.［Ch，6 fig. 3 tab. 15 ref.］