Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases

The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly.     

Development of a microarray for two rice subspecies: characterization and validation of gene expression in rice tissues

Background

Rice is one of the major crop species in the world helping to sustain approximately half of the global population’s diet especially in Asia. However, due to the impact of extreme climate change and global warming, rice crop production and yields may be adversely affected resulting in a world food crisis. Researchers have been keen to understand the effects of drought, temperature and other environmental stress factors on rice plant growth and development. Gene expression microarray technology represents a key strategy for the identification of genes and their associated expression patterns in response to stress. Here, we report on the development of the rice OneArray® microarray platform which is suitable for two major rice subspecies, japonica and indica.

Results

The rice OneArray® 60-mer, oligonucleotide microarray consists of a total of 21,179 probes covering 20,806 genes of japonica and 13,683 genes of indica. Through a validation study, total RNA isolated from rice shoots and roots were used for comparison of gene expression profiles via microarray examination. The results were submitted to NCBI’s Gene Expression Omnibus (GEO). Data can be found under the GEO accession number GSE50844 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50844). A list of significantly differentially expressed genes was generated; 438 shoot-specific genes were identified among 3,138 up-regulated genes, and 463 root-specific genes were found among 3,845 down-regulated genes. GO enrichment analysis demonstrates these results are in agreement with the known physiological processes of the different organs/tissues. Furthermore, qRT-PCR validation was performed on 66 genes, and found to significantly correlate with the microarray results (R?=?0.95, p?<?0.001***).

Conclusion

The rice OneArray® 22 K microarray, the first rice microarray, covering both japonica and indica subspecies was designed and validated in a comprehensive study of gene expression in rice tissues. The rice OneArray® microarray platform revealed high specificity and sensitivity. Additional information for the rice OneArray® microarray can be found at http://www.phalanx.com.tw/index.php.     

Vibrio nitrifigilis sp. nov., a marine nitrogen-fixing bacterium isolated from the lagoon sediment of an islet inside an atoll

A nitrogen-fixing isolate of facultatively anaerobic, marine bacterium, designated strain NFV-1^T, was recovered from the lagoon sediment of Dongsha Island, Taiwan. It was a Gram-negative rod which exhibited motility with monotrichous flagellation in broth cultures. The strain required NaCl for growth and grew optimally at about 25–35 °C, 3% NaCl and pH 7–8. It grew aerobically and could achieve anaerobic growth by fermenting d-glucose or other carbohydrates as substrates. NH₄Cl could serve as a sole nitrogen source for growth aerobically and anaerobically, whereas growth with N₂ as the sole nitrogen source was observed only under anaerobic conditions. Cellular fatty acids were predominated by C_16:1 ω7c, C_16:0, and C_18:1 ω7c. The major polar lipids consisted of phosphatidylethanolamine and phosphatidylserine. Strain NFV-1^T had a DNA G?+?C content of 42.5 mol%, as evaluated according to the chromosomal DNA sequencing data. Analyses of sequence similarities and phylogeny based on the 16S rRNA genes, together with the housekeeping genes, gyrB, ftsZ, mreB, topA and gapA, indicated that the strain formed a distinct species-level lineage in the genus Vibrio of the family Vibrionaceae. These phylogenetic data and those from genomic and phenotypic characterisations support the establishment of a novel Vibrio species, for which the name Vibrio nitrifigilis sp. nov. (type strain NFV-1^T?=?BCRC 81211^T?=?JCM 33628^T) is proposed.