Indigo dye is used to dye denim and other fabrics. It is now accepted that if this is co-extracted with the DNA, it may inhibit PCR amplification. A simple, improved method is described for the extraction of DNA from bloodstained denim for PCR amplification and short tandem repeat (STR) analysis. The DNA was extracted by constructing a blotting system using capillary action to draw a saline solution through the denim. The transferred material was collected onto nylon membranes and these were processed by chelex extraction. A variety of coloured denim substrates and other heavily dyed fabrics, including case work samples were used. In all cases the DNA was extracted, amplified and typed correctly.
The nucleotide sequence of a 4.39‐kb DNA fragment encoding the α‐glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the α‐glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N‐terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the α‐glucosidase precursor is proteolytically processed by removal of an N‐terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63–65 kDa and 50–52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expresion in the heterologus host, the C. tsukubaensisα‐glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensisα‐glucosidase, the rabbit sucrase‐isomaltase complex (proSI) and human lysosomal acid α‐glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.
The molecular cloning of an alpha-glucosidase gene isolated from a Candida tsukubaensis (CBS 6389) genomic library in Saccharomyces cervisiae is reported. The cloned gene is contained within a 6.2 kb Sau3A DNA fragment and directs the synthesis and secretion of an amylolytic enzyme into the extracellular medium of the recombinant host, S. cerevisiae. The cloned enzyme was found to have an unusually broad substrate specificity and is capable of hydrolysing alpha-1,2, alpha-1,3, alpha-1,4 and alpha-1,6 linked, as well as aryl and alkyl, D-glucosides. On the basis of its substrate specificity profile, the cloned enzyme was classified as an alpha-glucosidase (E.C. 3.2.1.20). It has a pH optimum in the range 4.2-4.6, a temperature optimum of 58 degrees C and is readily inactivated at pasteurization temperature (60 degrees C). Southern blot analysis failed to reveal any homology between the cloned gene and genomic DNA isolated from other well characterized amylolytic yeasts. A rapid plate-assay, based on the utilization of a chromogenic substrate X-alpha-D-glucoside to detect the expression of the cloned alpha-glucosidase in S. cerevisiae transformants, was developed.
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