Purpose

Identifying the sequence pattern in the radiation mutagenesis is essential for understanding the DNA molecular evolution. The present study aimed to establish an in vitro radiation mutagenesis model using naked DNA and to discover the sequence pattern of radiation-induced mutations.

Materials and methods

Human immunoglobulin heavy-chain variable region gene HVB was chosen as the targeted sequence. DNA molecules dissolved in water were irradiated with gamma rays (25Gy, 1 Gy/min), and four polymerases with different fidelity were used respectively for amplification. Ultra-sensitive next generation sequencing platform “EasyMF” was then employed to detect the mutation rate of all sites. Statistical analysis was performed using SAS9.4 with alpha 0.05.

Results

The main result of this study consisted of two parts. One was comparing of the effects of polymerase with different fidelity on ionizing radiation mutagenesis and establishing the in vitro model. Low fidelity DNA polymerases, Taq and Platinum® Taq DNA polymerase, could display the effects of radiation mutagenesis. However, due to the high error rate of Taq DNA polymerase, Platinum® Taq DNA polymerase was more suitable for studying the sequence pattern of radiation mutagenesis. Another was that mutation rates of individual nucleotides G, C and its doublet combination were significantly higher after radiation treatment.

Conclusion

Our findings suggest the fidelity of polymerase could affect the radiation mutagenesis. The nucleotides of G, C and their doublet combination showed higher radiation mutability. The sequence analysis of the radiation-induced mutation was helpful for the study of the evolution of DNA molecules under radiation. In addition, the comparisons of the polymerases was helpful to understand the role of the polymerase in the DNA damage repair.

中文翻译：

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使用新型体外模型进行γ射线诱变的序列偏好

目的

识别辐射诱变中的序列模式对于理解DNA分子进化至关重要。本研究旨在建立使用裸露的DNA的体外辐射诱变模型，并发现辐射诱导的突变的序列模式。

材料和方法

选择人免疫球蛋白重链可变区基因HVB作为靶序列。用γ射线（25Gy，1 Gy / min）照射溶解在水中的DNA分子，分别使用四种保真度不同的聚合酶进行扩增。然后使用超灵敏的下一代测序平台“ EasyMF”来检测所有位点的突变率。使用SAS9.4和alpha 0.05进行统计分析。

结果

这项研究的主要结果包括两个部分。一种是比较不同保真度的聚合酶对电离辐射诱变的影响，并建立体外模型。低保真DNA聚合酶Taq和Platinum®Taq DNA聚合酶可能会显示辐射诱变的作用。但是，由于Taq DNA聚合酶的错误率很高，Platinum®Taq DNA聚合酶更适合研究放射诱变的序列模式。另一个是放射治疗后，单个核苷酸G，C及其双链体组合的突变率明显更高。

结论

我们的发现表明聚合酶的保真度可能会影响辐射诱变。G，C和它们的双峰组合的核苷酸显示出更高的辐射变异性。辐射诱导的突变的序列分析有助于研究辐射下DNA分子的进化。另外，聚合酶的比较有助于理解聚合酶在DNA损伤修复中的作用。