Lamboy W F
Department of Horticultural Sciences, Cornell University, Geneva, New York 14456-0462, USA.
PCR Methods Appl. 1994 Aug;4(1):38-43. doi: 10.1101/gr.4.1.38.
The production of informative random amplified polymorphic DNA (RAPD) markers using PCR and a single primer is often accompanied by the generation of artifactual (noninformative) bands as well. When RAPD data are used to compute genetic similarity coefficients, these artifacts (false positives, false negatives, or both) can cause large biases in the numerical values of the coefficients. As a result, some workers have been reluctant to use RAPD markers in the estimation of genetic similarities. Artifactual bands are of two types: those caused by variation in experimental conditions, and those caused by characteristics of the DNA to be amplified. A procedure is described that allows for correction of the bias caused by the first type of artifact, providing that replicate DNA samples have been extracted, amplified, and scored. The resulting data are used to obtain an estimate of the proportion of false-positive and false-negatives bands. These values are then used to correct the bias in the computed similarity coefficients. Two examples are given, one in which bias correction is critical to the results, and one in which it is less important. The maximum percent bias, computed from the estimated proportions of false positives and false negatives in the RAPD data set, is proposed as a criterion for determining whether bias correction of the similarity coefficients is required or not. Although all reasonable efforts should be made to optimize PCR protocols to eliminate artifactual bands, when this is not possible, the methods described allow RAPD markers to compute genetic similarities reliably and accurately, even when artifactual bands resulting from variation in experimental conditions are present.
利用聚合酶链式反应（PCR）和单一引物生成信息性随机扩增多态性DNA（RAPD）标记时，常常也会伴随产生人为（无信息）条带。当使用RAPD数据计算遗传相似系数时，这些人为因素（假阳性、假阴性或两者皆有）会导致系数数值出现较大偏差。因此，一些研究人员不愿在遗传相似性估计中使用RAPD标记。人为条带有两种类型：由实验条件变化引起的条带，以及由待扩增DNA的特性引起的条带。本文描述了一种程序，前提是已提取、扩增并对重复的DNA样本进行评分，该程序可校正由第一种人为因素导致的偏差。所得数据用于估计假阳性和假阴性条带的比例。然后使用这些值校正计算出的相似系数中的偏差。给出了两个例子，一个例子中偏差校正对结果至关重要，另一个例子中其重要性较低。根据RAPD数据集中估计的假阳性和假阴性比例计算出的最大偏差百分比，被提议作为确定是否需要对相似系数进行偏差校正的标准。尽管应尽一切合理努力优化PCR方案以消除人为条带，但当无法做到这一点时，即使存在由实验条件变化导致的人为条带，本文所述方法也能使RAPD标记可靠且准确地计算遗传相似性。
