Detection of K-ras exon 1 mutations by constant denaturant capillary electrophoresis

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K-ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 M (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K-ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas. Copyright (C) 2004 John Wiley Sons, Ltd.

Applications of homemade kit in mutation detection of genes

Several methods of mutation detection, such as single-strand conformation polymorphism (SSCP), tandem SSCP/heteroduplex analysis and SNaPshot analysis were developed using homemade kit on ABI 310 genetic analyzer, and were successfully applied to mutation detection of 31 colorectal tumor samples. The sieving capability of homemade kit and commercial kit were compared, results demonstrate that homemade kit has higher resolution and shorter analysis time. In clinical tumor samples, 26% K-ras (exon 1) and 24% p53 (exons 7-8) were found to have mutations, and all mutations were single point variations. A majority of mutations occurred in one gene, only 1 tumor contained alterations in the two genes, which indicates that development of colorectal cancer lies on alternate pathways, and may correlate with different gene mutations.

Determination of urinary oxidative DNA damage marker 8-hydroxy-2 '-deoxyguanosine and the association with cigarette smoking

8-hydroxy-2'-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30 mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3 nM (signal-to-noise ratio S/N = 3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers (31.4 +/- 18.9 nM versus 14.4 +/- 7.6 nM, P = 0.0004; 23.5 +/- 21.3 mug g(-1) creatinine versus 12.6 +/- 13.2 mug g(-1) creatinine, P = 0.028). (C) 2004 Elsevier B.V. All rights reserved.

Analysis of sulfur-containing compounds in crude oils by comprehensive two-dimensional gas chromatography with sulfur chemiluminescence detection

This paper reports an analytical method for separating, identifying, and quantifying sulfur-containing compounds in crude oil fraction (IBP-360degreesC) samples based on comprehensive two-dimensional gas chromatography coupled with a sulfur chemiluminescence detector. Various sulfur-containing compounds and their groups were analyzed with one direct injection. 3620 peaks were detected including 1722 thiols/thioethers/ disulfides/1-ring thiophenes, 953 benzothiophenes, 704 dibenzothiophenes, and 241 benzonaphthothiophenes. The target sulfur compounds and their groups were identified based on the group separation feature and structured retention of comprehensive two-dimensional gas chromatography as well as standard substances. The quantitative analysis of major sulfur-containing compounds and total sulfur was based on the linear response of the sulfur chemiluminescence detector using the internal standard method. The sulfur contents of target sulfur compounds and their groups in 4 crude oil fractions were also determined. The recoveries for standard sulfur-containing compounds were in the range of 90-102%. The quantitative result of total sulfur in the Oman crude oil fraction sample was compared with those from ASTM D 4294 standard method (total S by X-ray fluorescence spectrometry), the relative deviation (RD%) was 4.2% and the precision of the method satisfactory.