Development and application of “Catching-whole-mutations-in-genome method" that aims at health promotion activity

[目的]开发一种新的检测已知和未知疾病相关基因突变的方法（“Catching-whole-mutations-in-genome method”; CWHG method）。[方法]用生物素化碳二亚胺或突变识别蛋白MutS检测热处理和缓慢冷却后形成的杂合子。以高脂血症相关的脂蛋白脂酶（LPL）基因为模型。[结果1：双链DNA（dsDNA）形成效率]在95℃下5 min后缓慢冷却处理DNA片段。用非变性凝胶检测dsDNA形成效率。当DNA浓度大于0.1pmolDNA/ul时，绝大多数样品被检测为双链DNA。[结果2：异源双链体形成的效率]使用4个和8个碱基的插入突变来促进异源双链体的检测。异源双链形成效率为40%。异源双链的最高效率为50%，因此该结果是令人满意的。[结果3：Re 关于我们 异源双链体与碳二亚胺的反应活性及DNA区域匹配增强剂的影响]异源双链体与碳二亚胺的反应特异性较低。虽然使用了DNA区域匹配的增强剂，但特异性没有提高。【结果四：异源双链体与MutS的反应性及匹配DNA区域增强剂的作用]异源双链体与MutS的反应性特异性高，但所有类型的异源双链体均不反应。DNA区域匹配的增强剂对这一点没有改善作用。G105 R突变在任何SSCP条件下均未检出，通过异源双链体与MutS之间的反应检测。【结果五：新发现的LPL基因突变]新突变的积累对于建立CWHG方法和高脂血症的早期诊断具有重要意义。在1例日本高脂血症患者中发现了LPL基因5 '上游内含子1和Y 61 X的一个新的大缺失（54 kb）。少

【Purpose】 Our aim is to develop a new method (“Catching-whole-mutations-in-genome method"; CWHG method) that can detect mutations of genes related to known and unknown diseases. 【Method】 Detection of heteroduplexs formed after heat treatment and slow cooling was carried out with biotinylated carbodiimide or mutation recognition protein MutS. Lipoprotein lipase (LPL) gene related to hypertriglyceridemia was used as a model. 【Result 1: Efficiency of double strand DNA (dsDNA)formation】 DNA fragment was treated with slow cooling after 5 min at 95℃. Efficiency of dsDNA formation was examined with nondenaturing gel. Most of the samples were detected as dsDNA in case of more than concentration of 0.1pmol DNA/ul. 【Result 2: Efficiency of heteroduplex formation】 Insertion mutations of four and eight bases were used to facilitate detection of heteroduplex. Efficiency of heteroduplex formation was 40%. This result was satisfactory, because maximal efficiency of heteroduplex was 50%. 【Result 3: Re … More activity between heterodupex and carbodiimide, and effect of reinforcing reagent of matching DNA region】 Specificity of reactivity between heteroduples and carbodiimide was low. Although reinforcing reagent of matching DNA region was used, the specificity was not improved. 【Result 4: Reactivity between heterodupex and MutS, and effect of reinforcing reagent of matching DNA region】 Specificity of reactivity between heteroduples and MutS was high, but all types of heteroduplex were not reacted. Reinforcing reagent of matching DNA region didn't improve this point. The G105R mutation, which was not detected at any SSCP conditions employed, was detected by reaction between heteroduplex and MutS. 【Result 5: Newly identified mutation in LPL gene】 The accumulation of new mutations is important for the establishment of CWHG method and early diagnosis of hypertriglyceridemia. Compound heterozygosity of a novel large deletion (54kb) from5' upstream region to intron 1 and Y61X in LPL gene was identified in a hypertriglyceridemic Japanese patient. Less