Randy P. Rasmussen

Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification

Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease i...

Calcium antagonist binding sites in failing and nonfailing human ventricular myocardium

Studies in myopathic hamsters have described an increase in calcium antagonist binding sites, which is presumably associated with an increase in the number of calcium channels. Such an abnormality might predispose the heart to further myocardial damage from calcium overload. We tested the hypothesis that calcium antagonist binding sites are increased in human idiopathic dilated cardiomyopathy by examining [3H]PN 200-110 and [3H]nitrendipine binding in membranes prepared from nonfailing controls and severely failing ventricles with idiopathic dilated cardiomyopathy. Despite the fact that beta receptor density was decreased by 50% in failing hearts (iodocyanopindolol Bmax 84.4 +/- 8.9 fmol/mg protein in nonfailing hearts vs 42.9 +/- 3.2 fmol/mg in failing hearts, P less than 0.01), dihydropyridine calcium antagonist binding sites were not reduced significantly by heart failure. Maximum binding of [3H]PN 200-110 was 92.9 +/- 19.4 fmol/mg protein in membranes derived from failing ventricles, and 93.5 +/- 17.4 fmol/mg in membranes derived from nonfailing ventricles (P = NS); values for [3H]nitrendipine maximum binding were similar to those for [3H]PN 200-110 and also were not reduced significantly in failing ventricles. Additionally, the dissociation constants (KD) for [3H]nitrendipine and [3H]PN 200-110 were not significantly different in failing and nonfailing heart. We conclude that dihydropyridine calcium antagonist binding sites are not altered significantly in the failing human left ventricle with idiopathic dilated cardiomyopathy.

Fluorescence Monitoring of Rapid Cycle PCR for Quantification

The polymerase chain reaction (PCR) benefits from rapid temperature cycling (Wittwer et al., 1994). In particular, rapid cycling appears to improve the quantitative PCR of rare transcripts (Tan and Weis, 1992). The glass capillaries used as sample containers for rapid cycling are natural cuvettes for fluorescence analysis. Fluorometric monitoring of PCR has been reported with double-stranded DNA (dsDNA) dyes (Higuchi et al., 1992; Higuchi et al., 1993; Ishiguro et al., 1995; Wittwer et al., 1997a) and sequence-specific probes (Lee et al., 1993; Livak et al., 1995; Wittwer et al., 1997a). We have integrated a fluorimeter with a rapid temperature cycler for fluorescence monitoring during amplification (Wittwer et al., 1997b). Both cycle-by-cycle fluorescence monitoring and continuous (within cycle) monitoring offer unique quantitative information.

Two Color Multiplexing and Typing of Human Papillomavirus Types 16,18 and 45 on LightCycler

Cervical cancer is the second leading cause of cancer in women worldwide [1,2]. Human Papillomaviruses (HPV) have been identified as the most important viral group associated with benign and malignant neoplasia in humans [3]. Several studies have demonstrated that infection with certain HPV types may progress over a period of years through the various stages of cervical intraepithelial neoplasia (CIN) to invasive squamous carcinoma [4,5,6]. There are more than 80 different HPV types that have been identified [7,8]. Currently, there are more than 20 HPV types that have been found linked to cervical cancer [9]. This group is further subdivided into three categories designated as low, medium and high to differentiate between the relative risk for developing cervical carcinoma from any particular HPV type infection [10]. From the high risk category HPV 16 ranks first followed by HPV 18 in being found at a higher frequency in CIN and cervical carcinomas [1,11]. As of today, HPV 45 is considered a medium/high risk type but there is supporting evidence indicating that HPV 45 follows HPV 18 in frequency and that it should be classified as a high risk type [1, 12].