Technique / Molecular Biology / PCR / Degenerate PCR

alpha-L-Arabinofuranosidases (AFases; EC 3.2.1.55) are exo-type enzymes, which hydrolyze terminal nonreducing arabinose residues from various polysaccharides such as arabinan and arabinoxylan. Genome-wide BLAST search showed that various bacterial strains possess the putative AFase genes with well-conserved motif sequences at the nucleotide and amino acid sequence levels. In this study, two sets of degenerate PCR primers were designed and tested to detect putative AFase genes, based on their three highly conserved amino acid blocks (PGGNFV, GNEMDG, and DEWNVW). Among 20 Bacillus-associated species, 13 species were revealed to have putative AFase genes in their genome and they share over 67% of amino acid identities with each other. Based on the partial sequence obtained from an isolate, an AFase from Geobacillus sp. was cloned and expressed in E. coli. Enzymatic characterization has verified that the resulting enzyme corresponds to a typical AFase. Accordingly, degenerate PCR primers developed in this work can be used for fast, easy, and specific detection and isolation of putative AFase genes from bacterial cells.

Isolation of disease resistance gene analogs (RGAs) using the conserved motifs of the resistance genes has attracted considerable attention since it was first reported more than a decade ago. In this study, RGAs are isolated using homology-based PCR to target the nucleotide binding site (NBS) conserved regions from hexaploid wheat varieties and a few accessions of wild types. Based on sequence similarity analysis, 83 of the sequenced clones were clustered as groups. Of these RGAs, 40 were in the NBS-LLR class, containing kinase-1a (GGVGKTT or GGVGKTA), kinase-2 (KRFLIVLDDXW), kinase-3a (GSXIVVITTR or GCXVLATTR), and the GLPL motif of the NBS-spanning region. Among these, 15 contained possible intron regions, similar to Avena sativa O2 NBS-LLR type disease resistance gene (AF078874), and one to Rpm1 of rice and Yr10 and Lr10 of wheat. To our knowledge, this is the first observation of an intronic site within the P-loop domain of wheat RGAs. We detected an unspecified motif (VMVCVS) between the kinase-1a and kinase-2 domains within our clones. Additionally, one of the clones showed replacement with the kinase-3a motif with an undefined sequence.

Expression of human endogenous retroviruses (HERV) has been recurrently observed during cellular differentiation or transformation processes in both cell culture and in vivo. Quantitative approaches that analyze variations in HERV transcription could therefore be valuable for cancer diagnosis. We have developed a quantitative assay combining multiplex degenerate PCR (MD-PCR) and a colorimetric Oligo Sorbent Array (OLISA). Quantification of the expression of these multifamily genes relies on the optimization of the amplification primer mix, that is, the primer degeneracy, the relative concentration of each primer and the total amount of primer. Amplification products of each of the nine studied HERV families are independently and specifically detected and quantified using the OLISA microarray. This method constitutes an improvement over previous pan-retrovirus amplification-based methods, which are mainly qualitative. Furthermore, as MD-PCR/OLISA simultaneously monitors several HERV families, it challenges single-family quantitative RT-PCR. Last, the protocol below provides general rules for the design of MD-PCR applications. Once primers have been designed and optimized, the procedure can be completed in 2 days.