The recovery of intact nucleic acids following agarose gel separation is an important and often-practiced procedure for the molecular biologist. It is necessary to obtain an uncontaminated sample of the nucleic acids of interest for successful cloning, restriction mapping, DNA sequencing, transformation of various vectors, microinjection, and amplification. There are many standardized protocols and kits that exist to facilitate this procedure. In particular, many of the popular purification kits employ spin column-silica gel membrane filtraton methods to purifiy nucleic acids following separation on agarose gels. However, on occasion, it may still be difficult to obtain the desired high yields from these procedures, as a significant percentage of DNA or RNA may be lost during the purification process. Also, the high temperatures required to melt agarose gels would denature double-stranded DNA and degrade RNA, rendering these purification schemes ineffective. Separating nucleic acids on low-melting point (LMP) agarose gels is a step further in enhancing nucleic acid purification and availability. While most standard agarose requires temperatures of ~ 85-95
oC to melt, LMP agarose melts at much lower temperatures (~65-70oC, staying molten at ~45oC), facilitating the recovery of nucleic acids (and avoiding the losses associated with purification from standard agarose gels). Still, purification from LMP agarose does not guarantee against low recovery yields, which may not sit well for someone already working with little to begin with. The addition of an agarose-digesting enzyme such as EPICENTRE’s GELase enzyme prep could further enhance the yield of nucleic acid recovery following LMP agarose gel separation, and also permit direct use of melted agarose gel-nucleic acid solution mixture in further cloning and amplification steps.
The GELase enzyme digests long-chain polysaccharides in molten agarose to give a clear liquid that does not solidify on cooling to 0oC. The gel slice may be digested either directly in standard electrophoresis buffers (i.e., TAE, TBE, MOPS) or in the provided GELase buffer. The digested liquid product is soluble in ethanol, permitting direct precipitation of nucleic acids from the solution without the need for phenol/chloroform extractions.
According to the instructions provided by EPICENTRE, LMP agarose digestion with GELase can be performed following a High Activity Protocol or a Fast Protocol. In the High Activity Protocol, a buffer exchange takes place prior to gel melting and digestion, in which electrophoresis buffer is replaced with the provided GELase buffer. This step is purported to ensure the highest levels of enzyme activity, so less enzyme can be used for each reaction. This is an important consideration if one has many reactions or is on a tight budget. Alternatively, in the Fast Protocol, the digestion is performed directly in electrophoresis buffer, following addition of GELase Buffer. This eliminates the buffer exchange step, and therefore saves an extra hour, but more enzyme is required. In general, 1 unit of GELase is reported to digest ~600 mg of 1% agarose in 1 hr at 45oC if using the High Activity Protocol, whereas 1 unit will digest less agarose in the same 1 hr using the Fast Protocol.
From my experience, I found GELase to moderately increase the yield of DNA purification from LMP agarose. In the instances when I've needed to keep nucleic acid losses to a minimum, I found GELase to be most useful, as I was able to bypass a purification step involving spin columns or phenol/chloroform extractions, and directly use the precious DNA-digested agarose mixture in further procedures such as ligation reactions. If faced with these similar circumstances, one should consider using GELase enzyme prep.
Joe Lau
Graduate Student
Mount Sinai School of Medicine, NY