Co‐precipitation of protein and polyester as a method to isolate high molecular weight DNA

摘要

The isolation of high molecular weight DNA is often achieved through extractions which involve the use of the hazardous organic solvents phenol and chloroform (Pellicer et al. 1978), adsorption protocols which employ chaotrophic salts such as GuSCN (guanidinium isothiocyanate) along with silica particles (Carter and Milton 1993), or by dehydration protocols which involve the addition of large amounts of NaCl (or other salts) in order to competitively precipitate proteins (salting out)(Miller et al. 1988). Each of these methods can produce high quality DNA and the salting out protocol is perhaps the safest method while the phenol:chloroform method reliably produces protein free, high quality DNA. The safety of the salting out protocol can be overshadowed by salt carryover when more salt is added than needed to precipitate cellular protein. Inversely, isolated DNA may be contaminated by protein if insufficient salt concentrations are used. Either of these conditions can result in inhibition of subsequent reactions which involve the isolated DNA.Polyethylene glycol is a widely used polymer for selective protein precipitation and acetone may also be used to precipitate cellular protein (Bollag et al. 1991; Ingham et al. 1984). Drawing from those applications, I envisioned that whole cell protein might be precipitated and immobilized by the addition of an acetone/monomeric ester resin solution followed by in vitro polymerization of the resin/protein complex, thus clearing a cellular lysate of protein while maintaining a nucleic acid solution consisting of oligomeric polymer, water, buffer and DNA. Presumably, longer strands of polyester will bind the protein fraction of a standard cell lysate and shorter oligoesters will bind DNA. The bound protein should precipitate quickly while the bound DNA remains suspended. The bound DNA can be precipitated and washed with ethanol and released from the oligoesters upon addition of water to the DNA/oligoester pellet. I have used the described method to isolate DNA from ～25 adipose fins and ～250 barbel samples from channel catfish (Ictalarus punctatus), and smaller numbers of skeletal muscle samples from largemouth bass (Micropterus salmoides). In the PCR, these isolated DNA samples amplified equal to or better than DNA obtained using the salting out protocol described by Miller et al. (Fig. 1) (Miller et al. 1988). Due to similarity among tissues from diverse organisms, this method will likely prove adequate for the isolation of DNA from tissues other than fish fin and skeletal muscle.Figure1. Open FigureDownload Powerpoint slideComparison of total DNA and a fragment amplified from total DNA which was isolated using a polyester co-precipitation technique. The DNA ladder on the left side of the image has the following bands (from bottom to top): 250 bp, 500 bp, 750 bp, 1?000 bp and 10?000 bp. The lanes of this composite gel include (A) DNA isolated from channel catfish adipose fin using the polyester method of DNA isolation, (B) DNA isolated from largemouth bass skeletal muscle using the polyester isolation method, (C) PCR product produced from DNA template isolated using the polyester method (D) PCR product produced from template DNA isolated by the salting-out method (Bollag et al. 1991). The high molecular weight of the isolated DNA, as well as its performance in the PCR, is a testament to the suitability of this method of DNA isolation for molecular techniques.