EXPERIMENTAL-ANALYSIS OF VARIANCE FOR DNA HYBRIDIZATION .1. ACCURACY

We used tissues of the Virginia opossum (Didelphis virginiana) to exam ine the experimental accuracy of DNA hybridization statistics of therm al stability (T(mode), T(m), T50H, and NPH) with respect to systematic biases in counting radioactivity in elution fractions, and column pos ition and loading order of hybrids in the thermal elution device. We f ailed to detect any change in the mean melting temperatures among five replicate I-125-labeled hybrids counted over 72 h. Furthermore, colum n position in the automated thermal elution device (TED) did not bias the statistics of aliquots loaded over a few minutes from a single lar ge ''mother'' hybrid. On the other hand, the normalized percentage hyb ridization (NPH) increased as much as 3-5% for aliquots loaded during 1 h from a similar ''mother'' hybrid. A parallel but less consistent i ncrease was noted for T50H, which incorporates a measure of NPH. This ''NPH effect'' disappeared when hybrids were prepared individually and diluted and loaded in turn-the usual procedure in our laboratory. Rep licate distances measured as NPH appear to be sensitive to departures from the normal-distribution assumption of least-squares regression. W e recommend that replicate cell values of NPH be transformed to improv e their fit to a normal distribution prior to analysis by least-square s phylogenetic algorithms such as those available in Felsenstein's PHY LIP package. Thus, potential sources of inaccuracy in DNA hybridizatio n data can be avoided with simple precautions.