Rojas F J, Garner C, Schiewe M, Asch R H, Balmaceda J P, Moretti-Rojas I
Department of Obstetrics and Gynecology, University of California, Irvine, Orange, USA.
Fertil Steril. 1995 Aug;64(2):255-60. doi: 10.1016/s0015-0282(16)57719-x.
To develop a simplified polymerase chain reaction (PCR) protocol on single cells for the purpose of preimplantation genetic diagnosis. Also to evaluate a new thermal cycler, RoboCycler 40 (Stratagene, La Jolla, CA), for reducing the time to complete PCR amplification.
PCR amplification without DNA purification or reamplification of a 149 base pair (bp) segment of the human Y chromosome was used as a model. The assay was tested in human fetal cells, single lymphocytes and single human blastomeres.
Amplification of the 149 bp segment using fetal cells was 100% correct. Results on single lymphocytes were concordant in all but one of the 15 male cases. However, 2 of the 25 female cases were identified as male suggesting the occurrence of DNA contamination. Analysis of 61 blastomeres were concordant in 57 cases (93%); results for male blastomeres showed 12% of false negatives. No false positives were detected for female cells. Amplification using the simplified PCR protocol in combination with the RoboCycler was completed in 2 hours.
These data show that this PCR assay performed directly, without DNA extraction or purification and without re-amplification is a practical and effective approach for amplification of specific DNA sequences in single cells. Furthermore, the simplified PCR protocol significantly reduced the time to complete DNA amplification. The reduced time is expected to facilitate the management of a routine program for preimplantation genetic diagnosis.
开发一种用于单细胞的简化聚合酶链反应（PCR）方案，以用于植入前基因诊断。同时评估一种新型热循环仪RoboCycler 40（Stratagene公司，加利福尼亚州拉霍亚），以缩短完成PCR扩增所需的时间。
以不进行DNA纯化或对人类Y染色体的149个碱基对（bp）片段进行再扩增的PCR扩增作为模型。该检测方法在人类胎儿细胞、单个淋巴细胞和单个人类卵裂球中进行测试。
使用胎儿细胞对149 bp片段的扩增100%正确。在15例男性病例中，除1例之外，单个淋巴细胞的检测结果均一致。然而，25例女性病例中有2例被鉴定为男性，提示存在DNA污染。对61个卵裂球的分析中，57例（93%）结果一致；男性卵裂球的检测结果显示有12%的假阴性。女性细胞未检测到假阳性。使用简化PCR方案结合RoboCycler进行的扩增在2小时内完成。
这些数据表明，这种直接进行、无需DNA提取或纯化且无需再扩增的PCR检测方法，是在单细胞中扩增特定DNA序列的一种实用且有效的方法。此外，简化的PCR方案显著缩短了完成DNA扩增所需的时间。预计时间的缩短将有助于管理植入前基因诊断的常规程序。
