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摘要: 模板DNA、引物、三磷酸碱基脱氧核苷酸（dNTPs），镁离子（Mg2+）浓度、Taq DNA聚合酶的用量以及退火温度是影响简单序列重复区间扩增?鄄聚合酶链式反应（ISSR-PCR）的主要因素。以麻楝Chukrasia tabularis叶片基因组DNA为试验材料，系统地测试这6个因素对麻楝ISSR-PCR反应结果的影响。结果表明：最优的反应体系为20 L反应体系中含30 ng模板DNA，1.00 molL－1随机引物，0.15 mmolL-1dNTPs，2.50 mmolL－1 Mg2+，2.50 16.67 nkat Taq DNA聚合酶。最佳退火温度为56 ℃，ISSR-PCR反应程序为94 ℃预变性5.0 min，94 ℃变性45 s，56 ℃退火45 s，72 ℃延伸1.5 min，40个循环；72 ℃再延伸7.0 min，4 ℃保存。应用优化的ISSR-PCR反应体系对24份麻楝个体材料进行扩增，均能扩增出丰富稳定的条带。图8表1参27Abstract: The genomic DNA from twenty-four varieties of germplasm material for Chukrasia tabularis leaves was systematically analyzed for template DNA，primer，dNTPs，Mg2+ concentration，dosage of Taq DNA polymerase，and annealing temperature with an inter simple sequence repeat（ISSR）-polymerase chain reaction（PCR）. Results showed that the optimal reaction system for the ISSR was a 20 L system containing 30 ng template DNA，1.00 molL－1 random primers, 0.15 mmolL－1 dNTPs，2.50 mmolL－1 Mg2+， and 2.50 16.67 nkat Taq DNA polymerase. The optimal annealing temperature was 56 ℃ with the PCR procedure pre-denaturized at 94 ℃ for 5.0 min, denaturized at 94 ℃ for 45 s, and annealed at 56 ℃ for 45 s with an extension at 72 ℃ for 1.5 min，a reaction of 40 cycles，and re-extension at 72 ℃ for 7 min. These products were then stored at 4 ℃. Results indicated that stable bands could be amplified. ［Ch，8 fig. 1 tab. 27 ref.］
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https://zlxb.zafu.edu.cn/article/doi/10.11833/j.issn.2095-0756.2013.04.024
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