人类颞骨火棉胶切片中线粒体DNA的扩增及重组测序

目的 应用分子生物学技术建立人类颞骨火棉胶切片中的ＤＮＡ分析方法。方法 采用多聚酶链反应（ＰＣＲ）配合不同的引物、扩增方式及酶切技术检测长期保存的７例（９侧）颞骨火棉胶切片中微量线粒体ＤＮＡ的片段缺失及点突变，ｐＧＥＭ－Ｔ载体进行扩增片段的重组与克隆。结果 ９侧颞骨ＤＮＡ提取液中均可见正常的１３５ｂｐ扩增片段，巢式ＰＣＲ检出其中２例生前患老年聋者（各查１侧）有线粒体ＤＮＡ大片段缺失，测序结果证实扩

Extraction, amplification, recombination and sequencing of the mitochondrial DNA from celloidin embedded human temporal bone sections]

OBJECTIVE: To establish an analytical method of DNA extracted from human celloidin-embedded temporal bone sections by molecular biologic technique. METHODS: Polymerase chain reaction (PCR) with different primers, special amplification methods or coorperated with enzyme restricted reactions was adapted to detect several types of genetic mutations from 9 human celloidin-embedded temporal bone sections. The pGEM-T vector was used for the recombination and cloning of the amplified DNA fragment. RESULTS: The 135 bp fragments of normal mtDNA were detected from all of the 9 cases. The 316bp fragment related to mtDNA 4977bp deletion was also detected in two cases who suffered from presbycusis before death by the nested PCR. The result of sequencing confirmed the accuracy of PCR. CONCLUSION: The application of molecular biologic techniques in temporal bone research is important in improving the quality and value of retrospective study of ear diseases and has resulted in findings of the relevant mutant or pathogenetic genes in the ear diseases such as presbycusis, ototoxic deafness, otosclerosis and viral infections of the ear.