摘要
用小规模提取法从海带“901”品系的配子体中提取基因组DNA,用于随机扩增多态DNA(RAPD)的重复性及稳定性研究。通过对扩增体系中各因子和扩增程序的梯度试验,确定其优化反应体系为:50 ng模板 DNA,2.0mmol/L MgCl_2,0.2mmol/L dNTP,0.2μmol/L引物,1.5 U Taq酶;所得 PCR反应程序为:94℃预变性 5min,45个循环:变性 94℃ 30 s,退火 36℃1min,延伸72℃ 2min,最后72℃延伸10min。本研究条件获得的RAPD图谱有较好的重复性和特异性,为深入研究海带“901”品系的遗传和变异提供了方法依据。
Genomic DNA was isolated from the gametophyte of Laminaria japonica '901' strain using improved small-scale method, and it exhibited reproducible and stable patterns with randomly amplified polymorphic DNA (RAPD) reaction. The ingredients and programs for amplified reactions were optimized respectively, and it resulted in 50 ng template DNA, 2. 0 mmol/L MgCl2 , 0. 2 mmol/L dNIP, 0. 2 μmol/L primer, and 1. 5 U/25μL Taq DNA polymerase. In addition, the optimized reaction condition concluded was that predenaturation 94℃, 5 min; 30 s 94℃ ; annealing 1 min 36 ℃; elongation 2 min, 72℃ ; 45 cycles; finally holding 10 min at 72℃ . Our testified methods could yield satisfactory RAPD patterns, and it provided methods for further studying the mechanism of inheritance and variation to Laminaria '901' strain.
出处
《海洋科学》
CAS
CSCD
北大核心
2003年第5期22-26,共5页
Marine Sciences
基金
国家高技术研究发展计划项目2001AA621090号
教育部留学回国资助计划课题资助
