title: A flexible and fully integrated system for amplification, detection and genotyping of genomic DNA targets based on microfluidic oligonucleotide arrays.
abstract: A strategy allowing for amplification, detection and genotyping of different genomic DNA targets in a single reaction container is described. The method makes use of primer-directed solution-phase amplification with integrated labeling in a closed, microfluidic oligonucleotide array. Selective array probes allow for subsequent detection and genotyping of generated amplicons by hybridization. The array contains up to 15,624 programmable features that can be designed, de novo synthesized and tested within 24 hours using an automated benchtop microarray synthesizer. This enables rapid prototyping and adaptation of the system to newly emerging targets such as pathogenic bacterial or viral subtypes. The system was evaluated by amplifying and detecting different loci of viral (HPV), bacterial (Bacillus sp.) and eukaryotic (human) genomes. Multiplex PCR and semi-quantitative detection with excellent detection limits of <100 target copies is hereby demonstrated. The high automation grade of the system reduces contamination risk and workload and should enhance safety and reproducibility.
authors.affiliation: Im Neuenheimer Feld, Heidelberg, Germany. daniel.summerer@febit.de
authors.forename: Daniel
authors.initials: D
authors.lastname: Summerer
authors.forename: Dona
authors.initials: D
authors.lastname: Hevroni
authors.forename: Amit
authors.initials: A
authors.lastname: Jain
authors.forename: Olga
authors.initials: O
authors.lastname: Oldenburger
authors.forename: Jefferson
authors.initials: J
authors.lastname: Parker
authors.forename: Anthony
authors.initials: A
authors.lastname: Caruso
authors.forename: Cord F
authors.initials: CF
authors.lastname: Stähler
authors.forename: Peer F
authors.initials: PF
authors.lastname: Stähler
authors.forename: Markus
authors.initials: M
authors.lastname: Beier
mesh_terms: D002874:Chromosome Mapping / Q000379:methods*; D004247:DNA / Q000235:genetics*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D018390:Gene Targeting / Q000295:instrumentation*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation*; D018511:Systems Integration
chemical_list: D004247:DNA