Walsh P S, Varlaro J, Reynolds R
Roche Molecular Systems, Alameda, CA 94501.
Nucleic Acids Res. 1992 Oct 11;20(19):5061-5. doi: 10.1093/nar/20.19.5061.
A sensitive and simple method for the quantitation of human DNA is described. This method is based on probe hybridization to a human alpha satellite locus, D17Z1. The biotinylated probe is hybridized to sample DNA immobilized on nylon membrane. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for chemiluminescent detection using a luminol-based reagent and X-ray film. Less than 150 pg of human DNA can easily be detected with a 15 minute exposure. The entire procedure can be performed in 1.5 hours. Microgram quantities of nonhuman DNA have been tested and the results indicate very high specificity for human DNA. The data on film can be scanned into a computer and a commercially available program can be used to create a standard curve where DNA quantity is plotted against the mean density of each slot blot signal. The methods described can also be applied to the very sensitive determination of quantity and quality (size) of DNA on Southern blots. The high sensitivity of this quantitation method requires the consumption of only a fraction of sample for analysis. Determination of DNA quantity is necessary for RFLP and many PCR-based tests where optimal results are obtained only with a relatively narrow range of DNA quantities. The specificity of this quantitation method for human DNA will be useful for the analysis of samples that may also contain bacterial or other non-human DNA, for example forensic evidence samples, ancient DNA samples, or clinical samples.
本文描述了一种灵敏且简便的人类DNA定量方法。该方法基于探针与人类α卫星位点D17Z1的杂交。生物素化探针与固定在尼龙膜上的样本DNA杂交。随后，链霉亲和素 - 辣根过氧化物酶与结合的探针结合，使用基于鲁米诺的试剂和X射线胶片进行化学发光检测。曝光15分钟即可轻松检测到少于150 pg的人类DNA。整个过程可在1.5小时内完成。已对微克量的非人类DNA进行了测试，结果表明该方法对人类DNA具有很高的特异性。胶片上的数据可扫描到计算机中，并使用市售程序创建标准曲线，将DNA量与每个狭缝印迹信号的平均密度进行绘图。所述方法也可应用于Southern印迹上DNA量和质量（大小）的非常灵敏的测定。这种定量方法的高灵敏度仅需消耗一小部分样本进行分析。在RFLP和许多基于PCR的测试中，确定DNA量是必要的，因为只有在相对较窄的DNA量范围内才能获得最佳结果。这种人类DNA定量方法的特异性对于分析可能还包含细菌或其他非人类DNA的样本将是有用的，例如法医证据样本、古代DNA样本或临床样本。
