Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography.

This work describes the application of the fully automated high-performance liquid chromatographic system to the analysis of PCR-amplified products. Efficient separations of both DNA restriction fragments and PCR products were performed using an anion-exchange DEAE-NPR column, packed with 2.5-microns nonporous particles. The automated HPLC method was employed for the separation, quantitation, and purification of PCR products in less than 10 min in a single step.

Quantitation and purification of polymerase chain reaction products by high-performance liquid chromatography.

The polymerase chain reaction (PCR) has rapidly become a standard laboratory technique. With the continuous development of PCR technology there is now a growing need for PCR product quantitation in areas such as therapeutic monitoring and quality control, disease diagnosis, and regulation of gene expression. One of the most common methods currently employed for post-PCR analysis is agarose or poly-acrylamide gel electrophoresis. However, the method is time-consuming and only semiquantitative. In contrast, high-performance liquid chromatography (HPLC) is well accepted as a quantitative technique in many diverse applications areas such as pharmaceutical, biotechnology, food, and environmental, since the technique can provide reliable, precise, and sensitive sample detection, and wide dynamic range.

Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.

The polymerase chain reaction (PCR), a new, powerful method for rapid enzymatic amplification of specific DNA fragments, has gained tremendous popularity in molecular biology. This paper describes the successful application of liquid chromatography to the analysis of products of the PCR. Efficient separation of both DNA restriction fragments and amplified PCR products were achieved in 10-12 min on a new ion-exchange column, DEAE-NPR, packed with 2.5-microns non-porous particles. The PCR products were quantitated with a reproducibility within 10%. Use of liquid chromatography was demonstrated for separation and quantitation of PCR products in amounts below those required for direct analysis by ethidium bromide gel electrophoresis or a Hoechst 33258 dye-based fluorescence assay. Liquid chromatography was also demonstrated to be effective for quick optimization of PCR procedures.

Rapid analysis and purification of polymerase chain reaction products by high-performance liquid chromatography.

This report describes the use of high-performance liquid chromatography (HPLC) for the rapid analysis and purification of the polymerase chain reaction products. Employing a new anion-exchange nonporous column, efficient separations of both DNA restriction fragments and amplified PCR products are achieved in 10 to 20 minutes and quantitated within +/- 10%. The performance of the HPLC technique is described in terms of resolution, reproductibility, sensitivity and micropreparative capability and compared to that of gel electrophoresis for this application.

Muscarinic actions and receptor binding of the enantiomers of BM 130, an alkylating analog of oxotremorine.

The enantiomers of the oxotremorine analog N-[4-(2-chloromethylpyrrolidine)-2-butynyl]-2-pyrrolidone (BM 130) were synthesized. The LD50 values of (+)- and (-)-BM 130 in mice (i.v.) were 10.4 +/- 1.4 and 13.5 +/- 1.9 mumol/kg, respectively. Atropine and N-methylatropine poorly protected against the lethal effects, suggesting that they were nonmuscarinic in nature. When administered i.v. to mice, (+)- and (-)-BM 130 were equipotent in producing peripheral and central muscarinic effects. ED50 values were 1.3 to 1.4, 2.8 to 3.2 and 0.20 to 0.26 mumol/kg, respectively, for salivation, tremor and analgesia (tail-flick assay). After i.p. injection, tremor was not observed and analgesic potency was reduced more than 10-fold compared to the i.v. route. The aziridinium ions [(+)- and (-)-BM 130A], formed by spontaneous cyclization of (+)- and (-)-BM 130, were virtually equipotent in eliciting contractions of the isolated guinea pig ileum and in causing salivation in mice. Their LD50 values in mice (i.v.) were 1.1 +/- 0.2 and 2.1 +/- 0.3 mumol/kg, respectively. The enantiomers of BM 130A had similar affinity for muscarinic receptors in the rat cerebral cortex as measured by competitive inhibition of (-)-[3H]N-methylscopolamine binding at 0 degrees C. The rate constants for alkylation of muscarinic receptors, obtained at 37 degrees C by measuring the decline in (-)-[3H]-3-quinuclidinyl benzilate binding to cortical homogenates that had been treated with various concentrations of (+)- and (-)-BM 130A for 20, 45 or 90 min, differed significantly for the two enantiomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylating partial muscarinic agonists related to oxotremorine. N-[4-[(2-haloethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidones.

N-[4-[(2-Chloroethyl)methylamino]-2-butynyl]-5-methyl-2-pyrrolidone (3) and N-[4-[(2-bromoethyl)methylamino]-2-butynyl]-5-methyl-2- pyrrolidone (4) were synthesized. Compounds 3 and 4 cyclized in neutral aqueous solution to an aziridinium ion (4A). The rate constants for the cyclization of 3 and 4 at 37 degrees C were 0.025 and 0.89 min-1, respectively. The aziridinium ion was equipotent with carbachol as a muscarinic agonist on the isolated guinea pig ileum. It was more potent than the corresponding 2-pyrrolidone derivative (2A) in alkylating muscarinic receptors in homogenates of the rat cerebral cortex. This higher potency was due to greater receptor affinity of 4A as compared to 2A rather than to greater rate constant for alkylation of muscarinic receptors. These properties of 3 and 4 and their low toxicity should make them valuable tools for receptor inactivation studies in vivo and in vitro.