快速鉴定苯丙酮尿症模型小鼠基因型的新方法应用  

Use of new methods of rapid genotyping the model mice for phenylketonuria

作　　者：贾兴元[1] 韩瀚[1] 肖白[1] 高斌[1] 周淑佩[2] 刘冕[2] 刘敬忠[1] 

机构地区：[1]首都医科大学附属北京朝阳医院基础医学研究中心,北京100020 [2]北京大学医学部实验动物中心,北京100083

出　　处：《诊断学理论与实践》2009年第6期631-634,共4页Journal of Diagnostics Concepts & Practice

基　　金：北京市重大专项"重大出生缺陷诊断及治疗研究"(D0906005040591)

摘　　要：目的:建立融解曲线法和变性高效液相色谱(DHPLC)法准确、快速地鉴定苯丙酮尿症(PKU)模型小鼠的基因型。方法:融解曲线法采用聚合酶链反应(PCR)扩增包含模型小鼠pah基因点突变位点序列的132bp片段,将该产物予Alw26I酶切后,加入微量SYBR Green I,在实时PCR仪上行融解曲线分析,同时,酶切产物行12%聚丙烯酰胺凝胶电泳分析。DHPLC法是直接将132bp片段扩增产物采用在部分变性条件下(60.9℃)行DHPLC分析,根据不同图谱作出基因型诊断。结果:融解曲线分析表明,野生型小鼠的DNA片段在84℃和81℃出现2个融解峰,突变纯合型小鼠DNA片段在76.5～77.5℃和81℃出现2个融解峰,杂合型小鼠DNA片段则在84℃、81℃和76.5～77.5℃均出现融解峰,3种峰型明显可被区分。DHPLC优化了最佳分离效果的柱温,在优化的最佳条件下,DHPLC图谱显示杂合型小鼠DNA具有显著特色的峰型,即除同源双链峰外,出现一个异源双链峰。上述2种新方法共检测了PKU模型小鼠突变纯合型20只、杂合型22只,野生型11只,2种方法检测结果一致,并且与经典的聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法结果一致(动物毛色等表型也完全一致)。结论:融解曲线法和DHPLC法均能准确、快速地鉴定模型小鼠的基因型,具有自动化、高通量等优点。Objective Establishing two methods for rapid and accurate identification of genotype of phenylketonuria model mouse. Methods A 132 bp fragment containing model mouse pah point mutation was amplified with PCR. The PCR products were digested with restriction enzyme Alw 26I and minute amount of SYBR Green I was added. Then the dissociation curve was analyzed on real-time PCR instrument; the digestion product was electrophoresized on 12% polyacrylamide gel meanwhile. The second technique analyzed the amplified 132 bp fragment by DHPLC （60.9℃） and the genotype was identified through profile patterns. Results The dissociation curve showed two peaks at 84 ℃ and 81℃ for wild type DNA,two peaks at 76.5-77.5℃ and 81℃ for homozygous genotype, and three peaks at 84℃,81℃ and 76.5-77.5 ℃ for heterozygous genotype. These three peak types could be distinguished easily. The results were in consistence with the results of PCR-RFLP. The column temperature was optimized for the DHPLC method.Under the optimum condition, the DHPLC profile pattern showed characteristic peak types： homoduplex peak and a heteroduplex peak. Twenty homozygous genotype mice,22 heterozygous genotype mice and 11 wild genotype mice were detected with these two new established methods. The results of these two methods were consistent with each other and with the classic PCR-RFLP. Conclusions Both the dissociation curve method and the DHPLC method can be used to identify the genotypes of phenylketonuria model mice with high accuracy, and the advantages of automation and high-influx.

关 键 词：实时聚合酶链反应 变性高效液相色谱 基因突变 检测 苯丙酮尿症模型小鼠 

分 类 号：R446.119[医药卫生—诊断学]