On-chip PCR amplification of very long templates using immobilized primers on glassy surfaces.
In this paper we describe a novel method for visualizing very long DNA fragments (for example >6 kb) which are difficult to spot with commonly used arrayers or capillary samplers with very small nanoliter volumes, using directly bound primers on "on-chip" polymerase chain reaction (PCR). We have used the genomes of the M13 bacteriophage (7.2 kb) the human mitochondrion (16.5 kb) as examples of long DNA templates to test the PCR and were able to elicit robust reactivity. Over 75% of the immobilized primers could be elongated to their fullest extent. In addition we were able to elicit the PCR reaction with double stranded templates in which one primer was immobilized and the other suspended in the reaction solution. These synthesized PCR products were visualized by either confocal microarray scanning or fluorescence microscopy using Cy5-dye fluorescence of the modified free primer, or the fluorescence of intercalating dyes.
von Nickisch-Rosenegk
Markus
M
Department of Molecular Bioanalysis and Bioelectronics, Fraunhofer Institute for Biomedical Engineering, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany. markus.nickisch@ibmt.fraunhofer.de
Marschan
Xenia
X
Andresen
Dennie
D
Abraham
Alexandra
A
Heise
Christian
C
Bier
Frank F
FF
D000327:Adsorption; D017104:Bacteriophage M13 / Q000235:genetics; D002874:Chromosome Mapping / Q000295:instrumentation / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005898:Glass / Q000737:chemistry*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013698:Templates, Genetic
D017931:DNA Primers; D004272:DNA, Mitochondrial; D004279:DNA, Viral