Department of Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
Biotechniques. 2010 Dec;49(6):893-7. doi: 10.2144/000113567.
Optimal accuracy of quantitative PCR (qPCR) requires correction for integrity of the target sequence. Here we combine the mathematics of the Poisson distribution and exponential amplification to show that the frequency of lesions per base (which prevent PCR amplification) can be derived from the slope of the regression line between cycle threshold (Ct) and amplicon length. We found that the amplifiable fraction (AF) of a target can be determined from this frequency and the target length. Experimental results from this method correlated with both the magnitude of a damaging agent and with other measures of DNA damage. Applying the method to a reference sequence, we determined the values for lesions/base in control samples, as well as in the AFs of the target sequence in qPCR samples collected from leukemic patients. The AFs used to calculate the final qPCR result were generally >0.5, but were <0.2 in a few samples, indicating significant degradation. We conclude that DNA damage is not always predictable; quantifying the DNA integrity of a sample and determining the AF of a specific qPCR target will improve the accuracy of qPCR and aid in the interpretation of negative results.
定量 PCR（qPCR）的最佳准确性需要校正目标序列的完整性。在这里，我们结合泊松分布和指数扩增的数学原理，表明每个碱基的病变频率（阻止 PCR 扩增）可以从循环阈值（Ct）和扩增子长度之间的回归线斜率推导出来。我们发现，目标的可扩增分数（AF）可以根据该频率和目标长度来确定。该方法的实验结果与致伤剂的大小以及 DNA 损伤的其他测量值相关。通过将该方法应用于参考序列，我们确定了对照样本中每碱基病变的数值，以及从白血病患者采集的 qPCR 样本中目标序列的 AF 值。用于计算最终 qPCR 结果的 AF 值通常大于 0.5，但在少数样本中小于 0.2，表明存在明显的降解。我们得出结论，DNA 损伤并非总是可预测的；量化样本的 DNA 完整性并确定特定 qPCR 靶标 AF 将提高 qPCR 的准确性，并有助于解释阴性结果。
