Guanidinium Salt Method

To extract DNA and RNA from serum samples or from culture supernatant fractions which contain a low level of proviral DNA, the IsoQuick extraction kit (a modified guanidinium salt-organic solvent extraction method) by MicroProbe Corporation (Bothell, WA) has been used successfully by our group. Following the manufacturer s recommended procedures for total nucleic acid extraction, the final DNA-RNA suspension can be amplified directly to detect proviral DNA. Using cDNA templates synthesized from total nucleic acids with random hexamer priming generally increases the PCR product yield. 

We introduced these guanidinium salts in a 1985 patent (Ref. 6) on the conversion of carboxylic acids to acid chlorides with phosgene. In this process, only 0.02 mol. % of HBGCI was required, two orders of magnitude less than the quantities of other catalysts typically used. Many new other applications including phosgene reactions with phenols, thiols, aldehydes, epoxides or O-demethyla-tion methods have been developed later and are discussed in this book. 

Brunelle DJ, Ye Q. Method for preparing oxy-diphthalic anhydrides using guanidinium salt as catalyst. US patent 6706897, assigned to General Electric Company, Niskayuna, NY 2004. 

If the protein of interest is a heteromultimer (composed of more than one type of polypeptide chain), then the protein must be dissociated and its component polypeptide subunits must be separated from one another and sequenced individually. Subunit associations in multimeric proteins are typically maintained solely by noncovalent forces, and therefore most multimeric proteins can usually be dissociated by exposure to pEI extremes, 8 M urea, 6 M guanidinium hydrochloride, or high salt concentrations. (All of these treatments disrupt polar interactions such as hydrogen bonds both within the protein molecule and between the protein and the aqueous solvent.) Once dissociated, the individual polypeptides can be isolated from one another on the basis of differences in size and/or charge. Occasionally, heteromultimers are linked together by interchain S—S bridges. In such instances, these cross-links must be cleaved prior to dissociation and isolation of the individual chains. The methods described under step 2 are applicable for this purpose. 

A series of reference proteins of known molecular masses are used to calibrate the column and Mr for an unknown protein is estimated from its position on the graph.195,196 Another modification of the method depends upon chromatography in a high concentration of the denaturing salt guanidinium chloride. The assumption is made that proteins are denatured into random coil conformations in this solvent.196... 

The hydration structures of several polyatomic ions such as NDJ, NO3, NO2, and CIO4 have already been established. Recently NDIS methods have also been applied to more complex ions such as Gdm+ and SCN (Fig. 4), which as the salt guanidinium thiocyanate is a particularly strong denaturant of proteins in solution, and both ions play leading roles in the Hofmeister series. ... 

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