title: LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin.
abstract: Oligonucleotide microarray has been one of the most powerful tools in the 'Post-Genome Era' for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.
authors.affiliation: Key Laboratory of Medical Cell Biology (Ministry of Education), Department of Biochemistry and Molecular Biology, First Affiliated Hospital, China Medical University, Shenyang, 110001, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Yan
authors.forename: Ying
authors.initials: Y
authors.lastname: Yuan
authors.forename: Runqing
authors.initials: R
authors.lastname: Mu
authors.forename: Hong
authors.initials: H
authors.lastname: Shang
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D001407:Bacillus / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D013696:Temperature
chemical_list: D015345:Oligonucleotide Probes