Circular dichroism and thermal melting of two small DNA restriction fragments of the same molecular weight.
The thermal melting and circular dichroism of two 147 base pair restriction fragments of pBR322 have been 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 two fragments are nearly equal. The circular dichroism spectra of fragments 12A and 12B in 0.2-10 mM Tris buffer are characteristic of B-form DNA. In 81% ethanol, the circular dichroism spectra of the two fragments are characteristic of A-form DNA. With 1 mM Tris buffer as the supporting electrolyte, fragment 12A exhibits a very sharp B----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.
D001482:Base Composition; D002942:Circular Dichroism; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial; D019003:Deoxyribonuclease HpaII; D006358:Hot Temperature; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D010957:Plasmids
D004269:DNA, Bacterial; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII