DNA循环测序中一些常见影响因素的研究

The influence of some factors on DNA cycle sequencing

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 factors on DNA cycle sequencing. Methods The effects of DNA templates, primers,cycle sequencing reaction conditions as well as purification methods were comparatively analyzed. Results When the DNA concentration was low, the nucleotide curve showed low ratio of signal to noise, even there were no fluorescent signals. While DNA concentration was too high,the readable length of nucleotides was short. Ions in the DNA template could result in bad sequencing reaction. The Tm value, length and G+C content of primers had no obvious influences on sequencing reaction.The alteration of the denaturation,annealing and extension temperature,or the addition of dimethl sulfoxide or glycerin facilitated sequencing some DNA templates. The fluorescent residues in the purified products of the sequence reaction could interfere the automatic reading of the sequencer,but did not influence the manually proofreading of the sequence. Conclusion The purity and concentration of DNA templates are closely related to sequence data quality. The modification of reaction parameters and usage of additives can help to obtain good result of sequencing some DNA with certain structure. The use of 70% ethanol is recommended to precipitate the extension product.