Pierce Kenneth E, Wangh Lawrence J
Department of Biology, Brandeis University, Waltham, MA, USA,
Methods Mol Biol. 2011;688:47-66. doi: 10.1007/978-1-60761-947-5_5.
Accurate detection of gene sequences in single cells is the ultimate challenge of PCR sensitivity. Unfortunately, commonly used conventional and real-time PCR techniques are often too unreliable at that level to provide the accuracy needed for clinical diagnosis. Here we provide details of Linear-After-The-Exponential-PCR (LATE-PCR), a method similar to asymmetric PCR in the use of primers at -different concentrations, but with novel design criteria to insure high efficiency and specificity. LATE-PCR increases the signal strength and allele discrimination capability of oligonucleotide probes such as molecular beacons and reduces variability among replicate samples. The analysis of real-time kinetics of LATE-PCR signals provides a means for improving the accuracy of single-cell genetic diagnosis.
准确检测单细胞中的基因序列是PCR灵敏度的终极挑战。不幸的是，常用的传统PCR和实时PCR技术在该水平上往往过于不可靠，无法提供临床诊断所需的准确性。在此，我们详细介绍指数后线性PCR（LATE-PCR），这是一种在使用不同浓度引物方面类似于不对称PCR的方法，但具有确保高效性和特异性的新颖设计标准。LATE-PCR提高了诸如分子信标等寡核苷酸探针的信号强度和等位基因区分能力，并降低了重复样本之间的变异性。对LATE-PCR信号的实时动力学分析为提高单细胞基因诊断的准确性提供了一种手段。
