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目的 研究 DNA循环测序中一些常见因素的影响。方法 对循环测序中模板、引物、测序反应条件及其产物的纯化等常见因素进行了比较研究。结果 测序模板浓度过低 ,则核苷酸曲线的信∶噪比小 ,甚至不出现荧光信号。模板浓度过高 ,判读的核苷酸长度降低 ;引物的长度、G+C含量及 Tm值对测序结果无明显影响 ;模板中的离子对测序反应有抑制作用 ;改变变性、退火及延伸温度 ,以及加入二甲基亚砜(DMSO)或甘油对某些 DNA模板的序列测定有较好的效果 ;测序反应产物纯化后如有残余的荧光染料 ,则会干扰测序仪对核苷酸的自动判读 ,但序列能进行人工校正。结论 序列质量与模板的纯度、浓度有明显的关系。对一些具有特殊结构 DNA,调整循环反应条件或使用添加剂能获得较好测序结果 ;利用 70 %乙醇沉淀法是首选的测序反应产物纯化方法。
Objective To study the influence of some common factors in DNA cycle sequencing. Methods The common factors such as template, primers, sequencing reaction conditions and the purification of their products in cycle sequencing were compared. Results Sequencing template concentration is too low, the letter of the nucleotide curve: noise ratio is small, or even no fluorescence signal. The length of the primer, the content of G + C and the Tm value had no significant effect on the sequencing results; the ions in the template inhibited the sequencing reaction; the denaturation, annealing and extension temperature were changed, As well as the addition of dimethyl sulfoxide (DMSO) or glycerol for sequencing of certain DNA templates. The residual fluorescent dye after purification of the sequencing reaction will interfere with the automatic interpretation of nucleotide sequences by the sequencer , But the sequence can be manually corrected. Conclusion The sequence quality has obvious relationship with the purity and concentration of the template. For some DNAs with special structure, better sequencing results can be obtained by adjusting the cycling reaction conditions or using additives; and the 70% ethanol precipitation method is the preferred purification method for sequencing reaction products.