Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly, the wild-type APECED protein tethered to the Gal4 DNA-binding domain acted as a strong transcriptional activator of reporter genes in mammalian cells, whereas most of the analyzed mutant polypeptides had lost this capacity.

Mutations in the AIRE gene: effects on subcellular location and transactivation function of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy protein.

Validation of the barcoding gene COI for use in forensic genetic species identification.

European species of Hypocrea Part I. The green-spored species

The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays and combinatorial chemistry. Such methods can include labeling a library of compounds by emulsifying aqueous solutions of the compounds and aqueous solutions of unique liquid labels on a microfluidic device, which includes a plurality of electrically addressable, channel bearing fluidic modules integrally arranged on a microfabricated substrate such that a continuous channel is provided for flow of immiscible fluids, whereby each compound is labeled with a unique liquid label, pooling the labeled emulsions, coalescing the labeled emulsions with emulsions containing a specific cell or enzyme, thereby forming a nanoreactor, screening the nanoreactors for a desirable reaction between the contents of the nanoreactor, and decoding the liquid label, thereby identifying a single compound from a library of compounds.

Microfluidic Devices And Methods Of Use In The Formation And Control Of Nanoreactors

Previously, an assay called conformation sensitive gel electrophoresis (CSGE) was developed for scanning PCR products for the presence of single-base and larger base mismatches in DNA. The assay was based on the assumption that mildly denaturing solvents in an appropriate buffer can accentuate the conformational changes produced by single-base mismatches in double-stranded DNA and thereby increase the differential migration in electrophoretic gels of heteroduplexes and homoduplexes. Here the sensitivity of assays by CSGE was improved by limiting the maximal size of the PCR products to 450 bp and making several changes in the conditions for PAGE. With the improved conditions, CSGE detected all 76 previously identified single-base changes in a large series of PCR products from collagen genes that contain multiple exons with highly repetitive and GC-rich sequences. In a survey of 736 alleles of collagen genes, CSGE detected 223 unique single-base mismatches that were confirmed by nucleotide sequencing. CSGE has the advantage over other methods for scanning PCR products in that it is simple, requires no special preparation of PCR products, has a large capacity, and does not use radioactivity.

https://www.pnas.org/content/pnas/95/4/1681.full.pdf

Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: Comparison with denaturing gradient gel electrophoresis and nucleotide sequencing

Structural alterations of the apolipoprotein E (apo E) are known to influence the lipid metabolism. The three most common isoproteins Arg 158) are encoded by three co-dominant alleles (e2, el, e4). Phenotyping is cumbersome and, therefore, several methods for apo E genotyping have been developed. Of these, PCR-RFLP (restriction fragment length polymorphism) is currently most rapid and cost-effective (1). To detect mutations not affecting restriction sites, direct PCR sequencing of apo E fragments is the method of choice. This, however, requires purification of PCR products, which is routinely accomplished by techniques like gel electrophoresis or chromatography. We have established a new purification method using exonuclease I (exo I) and shrimp alkaline phosphatase (sAP) to degrade excess primers and nucleotides, which are the main factors interfering with dideoxy PCR sequencing. This procedure simply requires the addition of the enzymes to the PCR product, avoiding frequent sample handling and DNA loss through technical manipulation. Human DNA was extracted from 50 iA EDTA blood with Tris-EDTA lysis and proteinase K digestion. The PCR reaction mix (50 ill) was optimized to contain 10 /tl of DNA extract, 30 pmol of the primers F4 and F6 (2), 5 pi of dimethyl sulfoxide (DMSO), 10 pmol of each dNTP, 2 U of Taq polymerase 5 ii\\ of 10 X PCR buffer (GeneAMP; Perkin-Elmer). After initial denaturation (95°C 5 min) 35 cycles (95°C 20 sec, 60°C 30 sec, 72 °C 30 sec) of amplification were performed, followed by a final extension (72°C 10 min; DNA Thermal Cycler 480, Perkin-Elmer). 2 jil of PCR product (244 bp) were mixed with 1 U exo I and 1 U sAP (USB, Cleveland, USA) and incubated for 1 h at 37°C. The enzymes were inactivated for 15 min at 72°C. The purified PCR product was diluted to a final volume of 10 /tl for cycle sequencing (3) with 0.5 pmol biotinylated primer F6 (A Taq™ Cycle Sequencing Kit; USB) under the same conditions as for PCR of genomic DNA. For chemiluminescence detection (4) we used the 'Sequenase® Images™ non-isotopic DNA sequence detection kit' (USB). Sequencing data obtained with sAP/exo I purified DNA are at least equal to those after column chromatography and superior to those using unpurified templates (Figure 1). Adjusting the purified DNA solutions for dilution we saw a slightly better efficiency of cycle sequencing in the enzymatically purified DNA.

Convenient single-step, one tube purification of PCR products for direct sequencing.

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Convenient single-step, one tube purification of PCR products for direct sequencing.