棉花微卫星DNA扩增产物检测方法的优化研究

Study on Optimum Method of Detecting Amplified Production of Microsatellite DNA in Upland Cotton

以棉花两个多标记基因系T582和T586及其后代为材料,对微卫星DNA的PCR扩增产物检测方法进行了优化研究。结果表明:检测微卫星DNA,聚丙烯酰胺银染灵敏度高于琼脂糖EB染色;聚丙烯酰胺凝胶的浓度需随着待测SSR序列的片段大小而作适当调整,一般情况下聚丙烯酰胺凝胶的浓度以6%为宜,但当待检测的DNA片段小到100bp～250bp的区域范围时,凝胶的浓度需提高到8%。胶板样品上样量以3滋l为宜。在显色液预冷(约10℃)的前提下,显色的时间应控制在4min之内,以便得到的胶板DNA条带强度适中、对比度好。

Multiple-marker lines in upland cotton, T582 and T586 and their hybrid progenies , were used as material. Detecting method of DNA polymorphism for cotton Microsatellite DNA was optimized in this study. The result showed that analysis of PCR amplified products of Microsatellite DNAs with silver-stained polypropylene gel was better than with ethidium-bromide-stained agarose gel. The concentration of polypropylene gel needs to be modified along with change of fragments size of DNA sequence. In general, 6% concentration of polypropylene gel was suitable. However, for the DNA fragments smaller than 100bp, 8% concentration of polypropylene gel was found better than 6%. Amount of 3?滋l amplified product added on gel wells was found enough. Developing band time should be limited within 4 min so as to make intension and clear if developing solution was in the cool condition.