生物发光分析法检测P53基因上的突变点

P53 GENE MUTATION DETECTION BY BIOLUMINOMETRY ASSAY

目的 建立简单、快速和廉价的基因突变测定方法。方法 采用基于生物发光技术的焦测序法测定基因突变 ,并通过毛细管和气压方式循环递加微量脱氧核糖核酸 (dNTP)完成焦测序反应。结果 建立了一种供焦测序反应的新型装置 ;研究了焦测序反应的影响因素和实验条件 ;实测了P5 3外显子 8上Cys2 75Ser突变点的 3种可能形态 (野生型、突变型和杂合型 ) ,并提出了快速判断基因突变类型的方法。结论 建立的方法简单 ,仪器装置成本低、操作简便 。

AIM To develop a simple, fast and inexpensive approach as well as an instrument for detection of gene mutation. METHODS Pyrosequencing based on bioluminometry assay was employed to detect gene mutation. Pyrosequencing is a method of sequencing by synthesis step by step using four enzymes, DNA polymerase, ATP sulfurylase, luciferase and apyrase. The signal was produced by detecting pyrophosphate released during a dNTP incorporation. For mutation detection, a DNA fragment was amplified by PCR at first, followed by a single stranded DNA preparation. In the second step, a short primer was annealed to the position just before the mutation point. Finally, specific dNTPs were added in terms of the template sequence. The mutation species can be readily determined by the sequence. RESULTS A new instrument was developed for gene mutation detection by pyrosequencing. To iteratively inject small amount of each dNTP for the sequencing reaction, capillaries were used to connect dNTP reservoirs and the reaction chamber. Each dNTP was delivered by adding a gas pressure on the top of a corresponding dNTP reservoir, by which 0 2 μL of dNTP can be exactly added each time. It was theoretically proved that undesired liquid seep through the capillary did not affect the sequencing reactions in pyrosequencing. In addition, the three possible variants (wildtype, mutant and heterozygote) of a mutant point Cys275Ser in P53 gene exon 8 were determined by pyrosequencing using the instrument. A simple method was also described for rapidly distinguishing the type of a variant. CONCLUSION The developed method is very simple, and the corresponding instrument is inexpensive and easy to operate, which can be used to detect many types of mutation.