改良测序聚丙烯酰胺凝胶和普通聚丙烯酰胺凝胶技术在玉米分子标记中的应用研究

为了快速、低成本获得高质量的玉米基因组DNA分子标记检测技术,在借鉴其他作物常规DNA分子标记检测的基础上,摸索了一套包括DNA提取及综合利用测序和普通聚丙烯酰胺凝胶电泳技术等DNA标记检测高效方法。以玉米为试验材料,提取其基因组DNA,选择不同玉米分子标记,并将标记扩增产物进行聚丙烯酰胺凝胶电泳,然后硝酸银染色,最终建立了一种应用于玉米分子标记的DNA提取和PAGE银染检测方法。在玉米DNA提取改良方面,本研究通过当天取玉米幼叶,立即利用电钻低温0⁴℃磨样,并减少1次抽提过程,结果显示提取DNA质量较好。在此基础上利用普通聚丙烯酰胺凝胶电泳检测PCR扩增结果,在优化部分参数后,检测效果依然较好;对其较难检测的差异过小的片段,通过改良的测序凝胶电泳检测,检测结果多态明显,且条带易读,灵敏度及分辨率均比普通聚丙烯酰胺凝胶电泳高。通过改良玉米DNA提取方法及综合利用测序和普通聚丙烯酰胺凝胶电泳技术,建立了一种行之有效、简便快捷的玉米分子标记应用研究技术。

Improved Big plate and Small plate of Polyacrylamide Gel Electrophoresis on Analysis of SSR Markers in Maize

In order to acquire the high quality of maize genome DNA molecular marker technology with fast and low cost,a set of DNA markers detection method including the DNA extraction and comprehensive utilization of sequencing and common polyacrylamide gel electrophoresis was explored on the basis of other crops of conventional DNA molecular marker detection. Using corn as test material,the genomic DNA of corn was extracted,and the products amplified by different markers were analyzed by polyacrylamide gel electrophoresis,and last stained with silver nitrate. Eventually PAGE silver staining method of a different marker in maize was established. On the improvement of extraction of DNA in Maize,the corn leaves were sampled and immediately grind using electric drill under the temperature of 0-4 degrees. One time of extraction was reduced. The results show that the quality of DNA is good. The improved common polyacrylamide gel electrophoresis method which simplifies operation steps,are less reagent dosage and no tail. The too small difference among of fragments which is hard to detect were detected by improved sequencing Polyacrylamide gel electrophoresis. It showed that the polymorphism is good and bands are clear and readable,of which sensitivity and resolution are higher than the ordinary polyacrylamide gel electrophoresis. By the improved DNA extraction and comprehensive application of the above two kinds of modified polyacrylamide gel electrophoresis method,an effective,simple and quick application of molecular marker techniques in Maize were established.