DNA循环测序中一些常见影响因素的研究

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

The influence of some factors on DNA cycle sequencing

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.