Andrea Frosini

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

ABSTRACT The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

Apolipoprotein E and Transferrin Genotyping by Ligation Detection Reaction and Universal Array

Genetic studies in Alzheimer disease (AD) have indicated that its etiology is multifactorial. The apolipoprotein E locus ( APOE ) is a known major susceptibility factor, and additional genetic loci have been associated with disease development (1)(2). Among many others, the transferrin gene ( TF ) has been suggested (3) as a candidate locus for AD because it is the major transport protein for iron, which itself is an important factor in free-radical generation. Oxidative stress and free-radical damage occur in AD, which justifies the interest in this protein. Previous studies have shown contrasting results regarding the influence of combinations of TF and APOE alleles (4)(5). We therefore designed a large-scale study of AD patients and controls to ascertain the relevance of TF as a risk factor for AD in conjunction with APOE . Here, we present the method that we have established for the simultaneous typing of the APOE and TF genes based on the ligation detection reaction (LDR)/universal array approach proposed by Gerry et al. (6). This method (Supplemental Fig. 1⇓ , A and B, accompanying the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue9/) is based on the PCR amplification of the regions including the polymorphic loci for TF and APOE genes. The resulting products are subjected to a multiplexed cycled ligation reaction that uses oligonucleotides designed to differentiate all possible alleles and that includes positive controls useful for normalizing the signals. This approach requires the design of a common LDR probe and two differentiating oligonucleotides for each polymorphic site. The common probe is phosphorylated on the 5′ end and contains a zip-code complement on its 3′-terminal …