Pierce Kenneth E, Rice John E, Sanchez J Aquiles, Wangh Lawrence J
Brandeis University, Waltham, MA 02454-9110, USA.
Biotechniques. 2002 May;32(5):1106-11. doi: 10.2144/02325pf01.
Amplification of DNA sequencesfrom single cells via PCR is increasingly used in basic research and clinical diagnostics but remains technically difficult. We have developed a cell lysis protocol that uses an optimized proteinase K solution, named QuantiLyse and permits reliable amplification from individual cells. This protocol was compared to other published methods by means of real-time PCR with molecular beacons. The results demonstrate that QuantiLyse treatment of single lymphocytes renders gene targets more availablefor amplification than other published proteinase K methods or lysis in water. QuantiLyse and an optimized alkaline lysis were equally effective in terms of target availability, although QuantiLyse offers greaterflexibility, as it does not require neutralization and can comprise a higher percentage of the final PCR volume. Maximum gene target availability is also obtained following QuantiLyse treatment of samples containing up to 10000 cells (the largest number tested). Thus, QuantiLyse maximizes the chances that targeted DNA sequences will be available for amplification during the first cycle of PCR, thereby reducing the variability among replicate reactions as well as the likelihood of amplification failure or allele drop-out. QuantiLyse will be useful in a range of investigations aimed at gene detection in small numbers of cells.
通过聚合酶链反应（PCR）对单细胞DNA序列进行扩增在基础研究和临床诊断中越来越常用，但在技术上仍具有挑战性。我们开发了一种细胞裂解方案，该方案使用一种优化的蛋白酶K溶液，名为QuantiLyse，可实现对单个细胞的可靠扩增。通过使用分子信标的实时PCR，将该方案与其他已发表的方法进行了比较。结果表明，与其他已发表的蛋白酶K方法或在水中裂解相比，用QuantiLyse处理单个淋巴细胞可使基因靶点更易于扩增。就靶点可用性而言，QuantiLyse和优化的碱性裂解同样有效，尽管QuantiLyse具有更大的灵活性，因为它不需要中和，并且可以在最终PCR体积中占更高的百分比。在用QuantiLyse处理含有多达10000个细胞（测试的最大数量）的样品后，也可获得最大的基因靶点可用性。因此，QuantiLyse可最大程度地提高在PCR第一个循环中靶向DNA序列可用于扩增的机会，从而减少重复反应之间的变异性以及扩增失败或等位基因缺失的可能性。QuantiLyse将有助于一系列旨在对少量细胞进行基因检测的研究。
