Resuspension of DNA sequencing reaction products in agarose increases sequence quality on an automated sequencer.

We are investigating approaches to increase DNA sequencing quality. Since a majorfactor in sequence generation is the cost of reagents and sample preparations, we have developed and optimized methods to sequence directly plasmid DNA isolated from alkaline lysis preparations. These methods remove the costly PCR and post-sequencing purification steps but can result in low sequence quality when using standard resuspension protocols on some sequencing platforms. This work outlines a simple, robust, and inexpensive resuspension protocol for DNA sequencing to correct this shortcoming. Resuspending the sequenced products in agarose before electrophoresis results in a substantial and reproducible increase in sequence quality and read length over resuspension in deionized water and has allowed us to use the aforementioned sample preparation methods to cut considerably the overall sequencing costs without sacrificing sequence quality. We demonstrate that resuspension of unpurified sequence products generated from template DNA isolated by a modified alkaline lysis technique in low concentrations of agarose yields a 384% improvement in sequence quality compared to resuspension in deionized water. Utilizing this protocol, we have produced more than 74,000 high-quality, long-read-length sequences from plasmid DNA template on the MegaBACET 1000 platform.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of riboflavin on the catalytic activity of liver fractions.

Weanling rats were kept on a synthetic riboflavin-free diet for 4 weeks, and subsequently on the same diet but supplemented with riboflavin for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. A decrease in both activities was evident during riboflavin deficiency, and this could be restored after normal supply of the vitamin. The decrease was attributed to a fall in the endogenous flavin content, specifically the coenzyme flavin adenine dinucleotide which forms an integral part of the microsomal monooxygenase that catalyzes the activation reactions. The vitamin and its coenzymes, however, inhibit the microsomal enzyme activity when added in excess in the in vitro system. It is envisaged that riboflavin may play a role in regulating the carcinogenic activity of AFB.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of copper on the catalytic activity of liver microsome.

Wealing rats were kept on a synthetic copper-free diet for 9-13 weeks and subsequently on the same diet but supplemented with copper at different levels for 2 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 leading to its activation and DNA adduct formation was measured after each period of experimental feeding. An increased molecular reactivity of aflatoxin B1 was evident during deficiency such that the degree of its conversion to its reactive metabolite and subsequent DNA adduct formation was higher with microsomes from copper deficient rats as compared to rats maintained on a normal supply of copper. These activities of microsomes returned to normal after supplementation of the diet with a small level of copper. The results emphasize the role of copper in the activation, and hence in modulating the carcinogenicity of aflatoxin B1.

In vivo effect of dietary factors on the molecular action of aflatoxin B1: role of vitamin A on the catalytic activity of liver fractions.

Weanling rats were kept on a synthetic vitamin A free diet for 9 weeks, and subsequently on the same diet but with oral supplementation of vitamin A palmitate at different doses for 2-3 weeks. The ability of liver microsomes to catalyze reactions of aflatoxin B1 (AFB1) leading to its activation and DNA adduct formation was measured after each period of experimental feeding. An increased molecular reactivity of AFB1 was evident during vitamin A deficiency such that the degree of its conversion to its reactive metabolite and subsequent DNA adduct formation was higher with microsomes from vitamin A-deficient rats as compared to rats maintained on normal supply of vitamin A. These activities of microsomes returned to normal after the rats had adequate supplements of vitamin A. The vitamin A status was also found to play a role in regulating glutathione S-transferase activity of the liver cytosol fraction, the activity being low in deficiency but increased progressively with increasing supplementation of vitamin A. Results suggest that vitamin A can afford protection against adverse effects of the potent hepatocarcinogen AFB.