摘要
目的 建立从人的单个细胞扩增特定靶基因片段进行基因诊断的技术。方法 利用显微操作技术挑取单个纤维母细胞 ,各置于 0 2ml簿壁反应管的裂解液中 ,合成针对抑癌基因P53第5～ 9外显子 (e)的引物 ,运用 1 5mer高度随机引物或一个特异引物进行单个细胞的整个基因组DNA或特异靶片段的预扩增 ,再以其为模板进行巢式聚合酶链反应 (PCR)扩增P53基因第e5～ 9的 1 90 0bp片段及含第 5 ,6外显子的 580bp片段。并用ABI 377测序仪测定其序列。结果 运用稀释至 0 0 1ng的基因组DNA扩增 1 90 0bp片段 ,优化PCR的条件。分别从 30个单个纤维母细胞扩增上述 1 90 0bp片段 ,1 0个获得 1 90 0bp片段 ,成功率为 33 %。扩增 580bp片段 ,则阳性率可达 60 %。序列分析证明确为人P53基因第 5 ,6外显子序列。结论 运用本实验室建立的技术可从人的单个细胞扩增特定靶基因片段 ,并测定其序列 。
Objective To found a technique of amplifying target gene fragments from single cells and optimize the conditions of polymerase chain reaction (PCR) Methods The single human fibroblast cells were obtained by using a microdissection instrument The whole genome sequences were amplified by using random 15 oligomer or/and one or more specific primers from the single cells of premer extension preamplification (PEP) Nest PCR was used to amplify a 1 900 bp P53 gene segment including exon 5～9 and a 580 bp segment including exon 5, 6 from the PEP product as template The 1 900 bp and 580 bp DNA fragments were sequenced by ABI 377 sequencer Results Optimized conditions of PEP and nest PCR for the 1 900 bp were obtained from diluted 0.01 ng genomic DNA that is equal to the DNA in one single cell. The 1 900 bp fragment was amplified from the single cell at a success rate of 33% The 580 bp fragment was amplified from the single cell at a much higer success rate that was about 60% Sequence analysis of the 1 900 bp and 580 bp fragment showed the same sequences as exon 5,6 of the P53 gene published Conclusion Target gene fragment from serveral hundrends bp to 1900 bp can be amplified and sequenced from single cells by using the method described in the article: The gene diagnosis could be made from the purified target DNA fragments from the single cells
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2002年第2期85-87,共3页
Chinese Journal of Laboratory Medicine
基金
北京市自然科学基金资助课题(JS96 0 0 4 )
