Use of normal probability paper in determining thermal melting values of deoxyribonucleic acid

Abstract

A method is described of plotting deoxyribonucleic acid (DNA) thermal melting data which allows information other than average content of guanine plus cytosine to be determined. Absorbancy (A) values, corrected for thermal expansion, are plotted on normal probability graph paper and the best straight line drawn. The 50 percentile point on the graph establishes the Tm value which allows calculation of the mean base composition. In addition the 2σ (twice the standard deviation) value is easily determined from the probability plot, thus providing information about the degree of variation from the mean in DNA base composition. Comparison of the probability plot and usual graphic methods of determining Tm values provided similar DNA composition data on 15 different strains of Streptococcus lactis, Streptococcus cremoris, and Streptococcus diacetilactis. When many A values obtained from streptococcal DNA samples showing skewed thermal melting curves (S. lactis b and S. diacetilactis DRC-2) were plotted on normal probability paper, two intersecting straight lines were apparent on the graph. This suggested that there were two types of DNA in these organisms. Each had its own unimodal compositional distribution and its presence was confirmed by density gradient separation. The use of normal probability plots as a rapid method of determining average base composition of DNA samples is suggested. In addition, heterogeneity in base composition may be shown by this method of plotting the data.