单淋巴细胞三重巢式PCR对dystrophin部分外显子和性别鉴定的实验研究

Single cell analysis of some deletion in dystrophin gene exons and gender determination by 3-plex nested PCR

目的 通过建立三重巢式PCR技术检测单个淋巴细胞的dystrophin基因和Y性别决定区(sex determiningregionY ,SRY)基因 ,探讨该技术对有家族史的缺失型Duchenne肌营养不良症 (Duchennemusculardystrophy ,DMD )家系中肯定携带者进行植入前遗传学诊断 ( preimplantation geneticdiagnosis ,PGD)临床应用的可行性。方法 在倒置显微镜下分别获取一名正常男性和一名正常女性的 5 0个单个淋巴细胞 ,用三重巢式PCR技术检测两组dystrophin基因和SRY基因 :外显子 5 1/外显子 19/SRY ,外显子48/外显子 19/SRY。结果 在外显子 5 1/外显子 19/SRY三重PCR反应体系中 ,正常男性第 5 1、19外显子及SRY的扩增率分别为 96%、94%和 94% ,正常女性第 5 1、19外显子扩增率分别为 94%、94% ;正常男女性假阳性率均为 6.7% ,假阴性率均为 0 ;在外显子 48/外显子 19/SRY三重PCR反应体系中 ,正常男性第 48、19外显子及SRY扩增率分别达 92 %、90 %和 94% ,假阳性率和假阴性率均为 0 ;正常女性第 48、19外显子扩增率分别达 94%和 92 % ,假阳性率和假阴性率分别为 6.7%和 0。结论 建立的三重巢式PCR技术具有较高的敏感性 ,可望在有家族史的缺失型DMD家系中进行PGD临床应用。

Objective To set up a technique of single lymphocytes 3-plex nested PCR for dystrophin and SRY gene, and to evaluate the possibility of using this technique for preimplantation genetic diagnosis(PGD) of deleted Duchenne muscular dystrophy(DMD)with family history. Methods Fifty single lymphocytes of a normal male and fifty of a normal female were obtained for detecting dystrophin gene(exon 51, exon 19, exon 48) and SRY gene by 3-plex nested PCR. Results In the group of exon 51/exon 19/SRY,the amplification rates of exon 51,exon 19 and SRY in male were 96%,94% and 94%; the amplification rates of exon 51 and exon19 in female were 94%and 94%, respectively. In the exon 48/exon 19/SRY group,the amplification rates of exon 48, exon 19 and SRY in male were 92%,90% and 94%, the amplification rates of exon 48, exon 19 in female were 94% and 92%, respectively. Conclusion The technique of single lymphocytes 3-plex nested PCR for dystrophin and SRY gene established in this study is highly sensitive,specific and reliable,and is suitable for PGD of deleted DMD with family history.