双错配碱基ARMS结合RE法快速检测FGFR3基因的突变超热点

目的建立快速简便、特异灵敏的鉴定FGFR3基因c．1138G〉A突变超热点的基因诊断方法，为软骨发育不全（ACH）的产前基因诊断或植入前遗传学诊断（PGD）创造条件。方法针对突变率高达95％以上的FGFR3基因的突变热点c．1138G〉A，设计双错配碱基的ARMS特异引物，对已经临床确诊的先证者家系及正常对照和非c．1138G〉A突变的患者对照，分别用普通引物和特异引物进行PCR扩增和直接鉴定，然后对扩增产物进行DNA序列分析及sfcI酶切鉴定。结果用普通引物扩增，对照组和先证者均扩增阳性。sfcI酶切后，对照组仍为一条513bp的带，而患者切出205bp、308bp和513bp三条带；而用ARMS特异引物扩增，则先证者扩增阳性，而对照组扩增阴性，阳性者酶切后产生27bp和418bp两条带。这一切都与预期的结果完全吻合。结论该法快速、特异、准确性高，结合DNA序列分析和酶切鉴定（RE），可用于ACH高危胎儿的快速产前诊断。

Rapid detection of FGFR3 gene mutation super hot spot with double mismatch base pairs ARMS combined with RE assay

Objective To establish a rapid, convenient, highly specific and highly sensitive diagnostic method to quickly determine FGFR3 gene c. 1138 G〉A super mutation hot spot, and provide a prerequisite for future prenatal gene diagnosis or preimplantation genetic diagnosis of achondroplasia. Methods For the FGFR3 gene mutation hot spot c. 1138 G〉A, whose mutation rate is up to 95%, we designed ARMS specific primer of double mismatch base pairs, PCR direct amplification and identification were performed on the pedigree of the clinical definite proband and normal controls and patient controls who weren＇ t c. 1138 G〉A mutation using ordinary primer and specific primer respectively, and DNA sequence analysis and SfcI enzyme digestion identification was conducted on the amplified products. Results The products of controls and proband, when using ordinary primer, are positive. As a result of SfcI enzyme digestion, controls showed a 513 bp band, while the patient had 205 bp, 308 bp and 513 bp bands. When using ARMS specific primer, the amplified production of proband is positive, and that of controls is negative, the positive one showed 27 bp and 418 bp bands after enzyme digested. All the results are fully in line with the expection. Conclusion The method is fast, accurate and highly specific. Combined with DNA sequence analysis and restriction enzyme digestion identification （RE）, this method can be applied to rapid prenatal diagnosis of ACH high risk fetals.