Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing.
Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of approximately 10(5)M(-1)s(-1) and dissociation rate constants of approximately 10(-4)s(-1). We found that by increasing the probe surface density from 10(11) to 10(12) molecules/cm(2), the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van't Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.
Philips Research, High Tech Campus, 5656 AE Eindhoven, The Netherlands. daniel.mocanu@infoworld.ro
Daniel
D
Mocanu
Aleksey
A
Kolesnychenko
Sonja
S
Aarts
Amanda T
AT
Dejong
Anke
A
Pierik
Wim
W
Coene
Erik
E
Vossenaar
Henk
H
Stapert
D001483:Base Sequence; D015342:DNA Probes / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D016373:DNA, Antisense / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008567:Membranes, Artificial; D009691:Nucleic Acid Denaturation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016062:Porosity; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics; D013212:Staphylococcus epidermidis / Q000235:genetics; D013816:Thermodynamics; D044366:Transition Temperature
D015342:DNA Probes; D016373:DNA, Antisense; D004269:DNA, Bacterial; D008567:Membranes, Artificial