DNA denatures upon drying after ethanol precipitation.
We have observed that ethanol precipitation and subsequent drying of small (less than 400 bp) radiolabelled DNA fragments is able to induce a transition to a form that migrates aberrantly on acrylamide gels. This unusual form has increased sensitivity to S1 nuclease, decreased sensitivity to restriction enzymes, and a concentration dependence for the reversion to the duplex form. Apparently, DNA denatures upon dehydration so that redissolving at low dilution will allow the collapse of DNA fragments into single-stranded hairpin structures. These structures are stable enough at low dilution to prevent complete reannealing of single stranded species. These single stranded species show strong binding to unidentified proteins present in nuclear extracts. This may give rise to misleading interpretations of mobility shift assays, especially if the single-stranded conformers have a similar mobility to the duplex fragment, which can occur in fragments that are 50-100 bp long. Evidence is presented that DNA, in general, denatures upon dehydration, but that hindrances to rotation in the solid state may prevent long fragments from dissociating.
Svaren
J
J
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 37232.
Inagami
S
S
Lovegren
E
E
Chalkley
R
R
D011232:Chemical Precipitation; D004247:DNA / Q000302:isolation & purification*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003890:Desiccation; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000431:Ethanol; D004247:DNA