Cawthon Richard M
Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA.
Nucleic Acids Res. 2009 Feb;37(3):e21. doi: 10.1093/nar/gkn1027. Epub 2009 Jan 7.
The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R(2) = 0.844) than with our original singleplex method (R(2) = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R(2) = 0.91).
当前用于测量端粒长度的定量聚合酶链反应（QPCR）分析，是在一组反应孔中测量实验DNA样本中的端粒（T）信号，并在单独的孔中测量单拷贝基因（S）信号，与参考DNA相比，以产生与平均端粒长度成比例的相对T/S比值。对该分析进行多重化是可取的，因为移液的DNA量的变化将不再导致T/S的变化，因为T和S将在每个反应中从相同的输入DNA中收集。多重化还提高了通量并降低了成本，因为所需的反应数量减少了一半。在这里，我们提出了第一种用于端粒长度测量的多重QPCR方法。值得注意的是，在这个系统中单一的荧光DNA嵌入染料就足够了，因为T信号可以在早期循环中收集，此时S信号尚未超过基线，而S信号可以在完全熔解端粒产物的温度下收集，使其信号回到基线。与我们原来的单重方法（R(2) = 0.677）相比，这种单色多重QPCR方法测得的T/S比值与通过Southern印迹法测量的末端限制片段（TRF）长度之间的相关性更强（R(2) = 0.844）。不同日期独立运行的多重T/S结果具有高度的可重复性（R(2) = 0.91）。
