ABSTRACT
Solid-phase techniques have facilitated the handling of biochemical analytes. This has stimulated the development of systems by which large sample panels can be analyzed with high levels of security and quality. We describe a sample transfer device based on the principle of vacuum filtration, which enables parallel handling of 96 samples of analytes bound to Sepharose beads. The tool was employed for strand separation of DNA samples, by attracting the beads to filter probes while passing them between the reagent solutions. The samples were analyzed using Pyrosequencing technology and proved to yield genotyping results of high quality. The presented sample preparation procedure provides an important link in the development of integrated systems for rapid genetic analysis at a low cost. In addition, the same filter could be reused extensively with very low risk for detectable cross-contamination between assays and without any reduction in processing capacity, thus further reducing the cost per analyzed sample.
Subject(s)
DNA/chemistry , Micromanipulation/instrumentation , Micromanipulation/methods , Sequence Analysis, DNA/methods , Specimen Handling/instrumentation , Specimen Handling/methods , Ultrafiltration/methods , DNA/genetics , Hot Temperature , Microspheres , Polymerase Chain Reaction/methods , Reproducibility of Results , Robotics/instrumentation , Robotics/methods , Sensitivity and Specificity , Sequence Analysis, DNA/instrumentation , VacuumABSTRACT
Fetal rat liver possesses substantial levels of glutathione S-transferase (GST) activity toward aflatoxin B1-8,9-epoxide. The enzyme responsible for this activity is an Alpha-class GST heterodimer comprising Yc1 and Yc2 subunits. The cDNAs encoding these polypeptides have been cloned and shown to share approximately 91% identity over 920 base pairs, extending from nucleotide -23 to the AATAAA polyadenylation signal. GST Yc2Yc2 expressed in Escherichia coli was found to exhibit 150-fold greater activity toward aflatoxin B1-8,9-epoxide than GST Yc1Yc1. Comparison between the structures of Alpha-class GST suggests that tyrosine at residue 108 and/or aspartate at residue 208 is responsible for the high aflatoxin B1 detoxication capacity of Yc2. Immunoblotting and enzyme assays have shown that liver from adult female rats contains about 10-fold greater levels of Yc2 than is found in liver from adult male rats. This sex-specific expression of Yc2 in adult rat liver may contribute to the relative insensitivity of female rats to aflatoxin B1. Dietary administration of oltipraz, a synthetic antioxidant which protects against aflatoxin-hepatocarcinogenesis, serves as an inducer of GST Yc2.
