Circular dichroism and thermal melting of two small DNA restriction fragments of the same molecular weight

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Abstract
The thermal melting and circular dichroism (CD), two 147 base pair restriction fragments of pBR322 were studied. The fragment with the higher GC content, 12B, melts at a higher temperature than the other fragment, 12A, as expected. The melting temperatures are proportional to the logarithm of the concentration of NaCl or tris(hydroxymethyl)aminomethane (Tris) buffer, between 1 mM and 0.2 M added salt. In 1 mM Tris buffer, the melting temperatures of the 2 fragments are nearly equal. The CD spectra of fragments 12A and 12B in 0.2-10 mM Tris buffer are characteristic of B-form DNA. In 81% ethanol, the CD of the 2 fragments are characteristic of A-form DNA. with 1 mM Tris buffer as the supporting electrolyte, fragment 12A exhibits a very sharp B .fwdarw. A transition, with a midpoint at 79% ethanol. However, a biphasic transition is observed for fragment 12B, with midpoints at 73 and 80% ethanol. This biphasic transition may represent the conversion of separate domains of fragment 12B from the B conformation to the A conformation; half of this fragment is much more GC rich than the other half. Methods are also described for preparing polymers of the 12A and 12B fragments.