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| 蚕微卫星DNA体外自我扩增条件初步研究 | |
| 引用本文： | 李关荣,廖芳,鲁成,向仲怀.蚕微卫星DNA体外自我扩增条件初步研究[J].蚕业科学,2003,29(3):231-235. |
| 作者姓名： | 李关荣 廖芳 鲁成 向仲怀 |
| 作者单位： | 1. 西南农业大学农学与生命科学学院,重庆,400716;西南农业大学蚕学与生物技术学院 2. 西南农业大学农学与生命科学学院,重庆,400716 3. 西南农业大学蚕学与生物技术学院 |
| 摘 要： | 采用一种类似PCR的基因组自我引发PCR(GSP PRC)法 ,初步研究了家蚕、野桑蚕、天蚕、蓖麻蚕和柞蚕的微卫星DNA的体外基因组自我引发PCR扩增条件。质量浓度为 10 0mg/L的基因组DNA经 10 0℃变性 15min后 ,与等量相同浓度未变性的DNA混合作为模板 ,在 80 μL扩增体系 含 0 2mmol/LdNTPs、5 0mmol/LKCl、10mmol/LTris HCl(pH 9 0 )、4mmol/LMgCl2 、1 2 5UTaqpolymerase]中加入 1μL此混合模板 ,在92℃、1min→ 5 5℃、2min→72℃、2min ,30～ 90次累积循环的扩增条件下 ,成功地得到了PCR产物。反应体系中 ,基因组DNA的适宜质量浓度为 1 2 5mg/L。GSP PCR产物经琼脂糖凝胶电泳后的部分片段回收后 ,在同样条件下 ,30～ 6 0个循环可得到同样大小范围的产物。GSP PCR产物经 6 %的变性测序胶电泳 ,得到典型的梯状带 ,表明为微卫星DNA。 |
| 关 键 词： | 蚕 微卫星DNA 基因组自我引发PCR |
| 文章编号： | 0257-4799(2003)03-0231-05 |
| 修稿时间： | 2003年2月10日 |
Preliminary Studies on the Conditions for the in vitro Self-amplification of Microsatellite DNA of Silkworm |
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| LI Guanrong , LIAO Fang LU Cheng XIANG Zhonghuai.Preliminary Studies on the Conditions for the in vitro Self-amplification of Microsatellite DNA of Silkworm[J].Acta Sericologica Sinica,2003,29(3):231-235. | |
| Authors: | LI Guanrong LIAO Fang LU Cheng XIANG Zhonghuai |
| Institution: | LI Guanrong 1,2 LIAO Fang 1 LU Cheng 2 XIANG Zhonghuai 2 |
| Abstract: | A PCR like genomic self priming PCR (GSP PCR) was applied in the preliminary study on the conditions for the preferential amplification of satellite DNA of silkworm. Genomic DNA at a concentration of 100 mg/L was heated at 100 ℃ for 15 min and mixed with an aliquot of unheated DNA of the same concentration. The mixed DNA was used as template for GSP PCR. 1 μL of this template was added into a 80 μL reaction containing 0 2 mmol/L dNTPs, 50 mmol/L KCl, 10 mmol/L Tris HCl (pH 9.0), 4 mmol/L MgCl 2, 1.25 U Taq polymerase] and amplified at 92 ℃ for 1 min, 55 ℃ for 2 min,72 ℃ for 2 min, 30 cycles. Depending on the results, products were re amplified 1～4 times by using 5 μL as template in a new reaction. GSP PCR products were successfully obtained. The suitable template concentration for GSP PCR was 1.25 mg/L reaction partial bands were recovered from agarose gel and re amplified under the same conditions; and the same partial bands were obtained. Sequencing gel electrophoresis revealed that GSP PCR products displayed a typical ladder type banding, indicating they were indeed micro satellite DNA. |
| Keywords: | SilkwormMicro satellite DNAGenomic self priming PCR(GSP PCR) |
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