Improved Silica-Guanidiniumthiocyanate DNA Isolation Procedure Based on Selective Binding of Bovine Alpha-Casein to Silica Particles

Abstract

DNA purified from clinical cerebrospinal fluid and urine specimens by a silica-guanidiniumthiocyanate procedure frequently contained an inhibitor(s) of DNA-processing enzymes which may have been introduced by the purification procedure itself. Inhibition could be relieved by the use of a novel lysis buffer containing alpha-casein. When the novel lysis buffer was used, alpha-casein was bound by the silica particles in the first step of the procedure and eluted together with DNA in the last step, after which it exerted its beneficial effects for DNA-processing enzymes. In the present study we have compared the novel lysis buffer with the previously described lysis buffer with respect to double-stranded DNA yield (which was nearly 100%) and the performance of DNA-processing enzymes.

Magnetic particles for the separation and purification of nucleic acids

“…Sorption processes based on (a) hydrogen-binding interaction with an underivatised hydrophilic matrix, typically silica, under chaotropic conditions, (b) ionic exchange under aqueous conditions by means of an anion exchanger, (c) affinity and (d) size exclusion mechanisms were used for DNA purification. Solid-phase systems which adsorb DNA-silicabased particles (Vogelstein and Gillespie 1979;Boom et al 1990Boom et al , 1999Melzak et al 1996;Tian et al 2000;Breadmore et al 2003), glass fibres, and anion-exchange carriers (Ferreira et al 2000;Endres et al 2003;Teeters et al 2003)-are used in chromatographic separation columns [e.g. DE 41 43 639 C2 (Qiagen GmbH)] for example.…”

Molecular Detection of Multiple Respiratory Viruses

“…In 1999, Boom and colleagues reported an extraction method based on reversible binding of nucleic acid molecules onto silica in the presence of chaotropic salts (for example, guanidine thiocyanate). This method removes endogenous nucleases and PCR inhibitors and purifies nucleic acid from various types of clinical samples (Boom et al, 1999). Most commercially available extraction kits and automated extraction platforms are based on this method.…”

Transfer of cholera toxin genes from O1 to non‐O1/O139 strains by vibriophages from California coastal waters

“…The phage pellets were resuspended in approx. 200 ll of the supernatant, and 100 ll was used for nucleic acid purification as previously described (Boom et al 1999). Two restriction enzymes, AluI (New England Biolabs, Ipswich, MA, USA) and EcoRI (Promega Corp., USA), were used for phage genome digestion following manufacturer instructions.…”

“…The primers and PCR condition were as previously reported (Rivera et al 2001;Jiang et al 2003). All cells were cultured in LB broth and genome extracted using guanidinium thiocyanate lysis and silica beads as previously described (Boom et al 1999). The positive amplification of zot gene from transductants (determined by 2% gel electrophoresis at 100 V for 1 h) indicated the transfer of zot gene.…”