Miyaguchi Hajime
National Research Institute of Police Science;
J Vis Exp. 2016 Sep 20(115):54402. doi: 10.3791/54402.
An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 ± 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.
本文描述了一种改进的聚合酶链反应（PCR）-限制性片段长度多态性（RFLP）方法，用于通过液相色谱/电喷雾电离质谱（LC/ESI-MS）对有毒河豚物种进行基因分型。使用基于硅胶膜的DNA提取试剂盒进行DNA提取。在使用无洗涤剂的PCR缓冲液进行PCR扩增后，无需纯化反应溶液，直接向溶液中加入限制性内切酶。分别采用反相硅胶整体柱和配备改进型金顿阱分析仪的傅里叶变换高分辨率质谱仪进行分离和检测。流动相由400 mM 1,1,1,3,3,3-六氟-2-丙醇、15 mM三乙胺（pH 7.9）和甲醇组成，以0.4 ml/min的流速输送。LC/ESI-MS分析的循环时间为8分钟，包括柱平衡时间。使用具有寡核苷酸同位素分布模型的去卷积软件从质谱图中计算相应的单同位素质量。对于寡核苷酸（范围为26-79个核苷酸）的分析，质量准确度为0.62±0.74 ppm（n = 280），并且在不使用锁定质量标准的情况下，180小时内保持了优异的准确度和精密度。
