细胞裂解法对植入前诊断中聚合酶链反应扩增效率的影响

Comparison of different lysis buffers in the assessment of the efficiency of single cell polymerase chain reaction for preimplantation genetic diagnosis

目的 :探讨优化细胞裂解条件 ,提高植入前诊断中聚合酶链反应的扩增效率。方法 :吸取单个淋巴细胞或人胚胎单卵裂球 ,用碱裂解液、蛋白酶K裂解液和改良的蛋白酶K裂解液进行处理 ,随后以巢式 -聚合酶链反应扩增人肾上腺脑白质营养不良基因 (ALD基因 )的 3号外显子 ,比较其扩增效率。结果 :三种裂解液组对人淋巴细胞的扩增效率分别为 90 % ,74 % ,87% ,对人单卵裂球的扩增效率分别为 98% ,77% ,96 %。结论 :在植入前诊断中 。

Objective To set up a highly efficient cell lysis system to increase the efficiency of single cell polymerase chain reaction (PCR) for preimplantation genetic diagnosis (PGD). Methods First we picked up one single lymphocyte or blastomere and lysed it in alkaline lysis buffer (ALB), proteinase K buffer (PK) and a new proteinase K lysis buffer compounded by ourselves. Then we amplified the 3rd exon of Gene ALD using nested PCR and compared the amplification efficiencies.Results The amplification efficiencies of the single lymphocyte in ALB, PK and the lysis buffer compounded by ourselves were 90%, 74%, and 87%; those of the single blastomere were 98%,77%, and 96%. Conclusion ALB and the lysis buffer compounded by ourselves are superior to PK in the lysis of cells in clinical PGD.