Prevention of depurination during elution facilitates the reamplification of DNA from differential display gels.
DNA fragments that show a pattern of differential expression on differential display gels must be eluted from the gel matrix and reamplified to enable further analysis. Elution is usually achieved by heating excised gel slices in a small volume of either water or TE. Here we show that this elution step can adversely affect the ability of the eluted DNA to act as a template for PCR reamplification, probably via the process of depurination. Simply switching to an elution solvent designed to minimise depurination (PCR buffer) facilitates the elution of intact DNA fragments. This improvement is likely to be most beneficial when eluting higher molecular weight fragments (e.g. those >500 bp), in situations where the amount of DNA in an excised gel slice is limited or when contaminating differential display products co-migrate with the differentially expressed product.
Frost
M R
MR
Department of Optometry and Vision Sciences, Cardiff University, Cardiff CF13 FX, UK.
Guggenheim
J A
JA
D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011687:Purines / Q000737:chemistry; D013696:Temperature; D013698:Templates, Genetic
D011687:Purines; D004247:DNA