DNA denaturation under freezing in alkaline medium.
It is generally accepted that DNA conserves its secondary structure after a freeze-thaw cycle. A negligible amount of degradation occurs after this procedure. Degradation becomes appreciable only after multiple cycles of freezing and thawing. In this study, we have found that a single freeze-thaw cycle in alkaline medium (pH>or=10.8) gives rise to denaturation of calf thymus DNA, although the melting temperature of intact DNA in the solution used for the freeze-thaw experiments is higher than 60 degrees C. The degree of denaturation is almost independent of the regime of freezing. The melting curve obtained after DNA is frozen at -2 degrees C and then thawed is almost the same as after a freezing carried out in liquid nitrogen (-196 degrees C). However, incubation in the same solution at 0 degrees C for 24 hours without freezing does not give rise to any denaturation. The degree of denaturation caused by freezing increases with pH (if pH>or=10.8) and decreases with Na2CO3 concentration at fixed pH and [Na+], although Na2CO3 decreases the melting temperature of intact DNA. A preliminary treatment of DNA with cisplatin or transplatin (0.01 Pt atoms per nucleotide) gives rise to a full recovery of the DNA secondary structure after freezing and thawing similar to what occurs after heating DNA to 100 degrees C and quick cooling. Possible mechanisms that may cause DNA denaturation during a freeze-thaw cycle in alkaline medium are discussed.
Galyuk
Elena N
EN
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, 5/2, Kuprevich St., 220141, Minsk, Belarus.
Wartell
Roger M
RM
Dosin
Yury M
YM
Lando
Dmitri Y
DY
D002945:Cisplatin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D015925:Cryopreservation; D004247:DNA / Q000737:chemistry*; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D017693:Sodium Bicarbonate / Q000737:chemistry; D044366:Transition Temperature
D003432:Cross-Linking Reagents; C034697:transplatin; D017693:Sodium Bicarbonate; D004247:DNA; C427203:calf thymus DNA; D002945:Cisplatin