Khan M A, Latif N, Petrobelli P, Yacoub M H, Dunn M J
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Heart Science Centre, Harefield Hospital, Middlesex, UK.
Electrophoresis. 1996 Jan;17(1):40-3. doi: 10.1002/elps.1150170107.
We describe a simple and effective method for generating competitive cDNA fragments for use as an internal standard in semi-quantitative polymerase chain reaction (PCR). The use of a nested composite primer to a region slightly within the expected amplicon but with heat shock protein 60 kDa (hsp60) primer sequence appended to the 5' terminus of the primer produces a "mimic" DNA that will model the PCR kinetics of the target template in sequence context, and PCR primer site, and that will be similar in size. This ensures that both competitor and target template are subjected to similar PCR kinetics and so allows more meaningful quantitation.
我们描述了一种简单有效的方法，用于生成竞争性互补DNA（cDNA）片段，以用作半定量聚合酶链反应（PCR）中的内标。使用嵌套复合引物，该引物针对预期扩增子内稍靠后的区域，但在引物的5'末端附加了热休克蛋白60 kDa（hsp60）引物序列，从而产生一种“模拟”DNA，该DNA将在序列背景、PCR引物位点方面模拟目标模板的PCR动力学，并且大小相似。这确保了竞争物和目标模板都经历相似的PCR动力学，从而实现更有意义的定量分析。
