Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that allows quantitative amplification detection at higher multiplexing by the integration of amplification in solution and monitoring via hybridization to a microarray in real-time. This method does not require any manipulation of the PCR product and runs in a single closed chamber. Employing labeled primers, one of the main challenges is to measure surface signals against a high fluorescence background from solution. A compact, confocal scanner is employed, based on miniaturized optics from DVD technology and combined with a flat thermocycler for simultaneous scanning and heating. The feasibility of this method is demonstrated in singleplex with an analytical sensitivity comparable to routine qrtPCR.

Rapid genotyping of human papillomavirus by post-PCR array-based hybridization techniques.

Kinetic hybridization measurements on a microarray are expected to become a valuable tool for genotyping applications. A method has been developed that enables kinetic hybridization measurements of PCR products on a low-density microarray. This is accomplished by pumping a solution containing PCR products up and down through a porous microarray substrate. After every pumping cycle, the fluorescently labeled PCR products hybridized to capture probes immobilized on the solid surface of the porous microarray substrate are measured. By this method, both binding curves and high-resolution melting curves are obtained instead of the single endpoint hybridization intensities as with commonly used post-PCR array-based hybridization techniques. We used 20 subtypes of the human papillomavirus (HPV) as a model system to test our detection method and blindly analyzed 216 clinical samples. We compared our microarray flowthrough method with a reference method, PCR followed by a reverse line blot (RLB). Real-time hybridization measurements followed by high-resolution melting curves of low concentrations of fluorescently labeled HPV targets on a microarray were successfully carried out without any additional chemical signal amplification. The results of our new method were in good agreement (93%, with a kappa coefficient of κ = 0.88 [95% CI, 0.81 to 0.94]) with the RLB results. All discrepant samples were analyzed by a third method, enzyme immunoassay (EIA). Furthermore, in a number of cases, we were able to identify false-positive samples by making use of the information contained in the kinetic binding and melting curves. This clearly demonstrates the added value of the use of kinetic measurements and high-resolution melting curves, especially for highly homologous targets.

Immobilization of oligonucleotides with homo-oligomer tails onto amine-functionalized solid substrates and the effects on hybridization.

Microarrays have become important tools for the detection and analysis of nucleic acid sequences. Photochemical (254 nm UV) DNA immobilization onto amine-functionalized substrates is often used in microarray fabrication and Southern blots, although details of this process and their effects on DNA functionality are not well understood. By using Cy5-labeled model oligonucleotides for UV immobilization and Cy3-labeled complementary sequences for hybridization, we measured independently the number of immobilized and hybridized oligonucleotides on the microarray surface. By using a two-color fluorescence LED setup and a novel method to compile the data, a full analysis has been made of the effects of oligonucleotide composition (length and sequence) on both immobilization and hybridization. Short homo-oligomer sequences (tails) of uracils, thymines, and, to a limited extent, guanines attached to a hybridization sequence improve immobilization. We propose a possible mechanism explaining the grafting of these nucleotides to amine-functionalized substrates, and we found evidence that the DNA backbone is possibly involved in the immobilization process. Hybridization, on the other hand, greatly improves as a function of tail length regardless of tail composition. On the basis of statistical arguments, the probes increasingly bind via their tail, with the hybridization sequence becoming more accessible to its complement. We conclude that all tails, sequence independent, improve hybridization signals, which is caused by either improved immobilization (especially thymine and uracil) or improved hybridization (most pronounced with guanine tails).

Mass transfer effects on DNA hybridization in a flow-through microarray.

The goal of this study is to assess the influence of mass transfer phenomena on DNA hybridization kinetics in a flow-through, porous microarray for fast molecular testing. We present a scaled mathematical model of coupled convection, diffusion and reaction in porous media, which was used to simulate hybridization kinetics and to analyze the influence of convective transport on the reaction rate. In addition to computer simulations, we also present experimental data of hybridization collected on our microarray system for different flow rates. The results reported in this paper provide for a better understanding of the interaction between reaction and mass transfer processes during flow-through hybridization and suggest criteria for system design and optimization.

Quality control of inkjet technology for DNA microarray fabrication.

A robust manufacturing process is essential to make high-quality DNA microarrays, especially for use in diagnostic tests. We investigated different failure modes of the inkjet printing process used to manufacture low-density microarrays. A single nozzle inkjet spotter was provided with two optical imaging systems, monitoring in real time the flight path of every droplet. If a droplet emission failure is detected, the printing process is automatically stopped. We analyzed over 1.3 million droplets. This information was used to investigate the performance of the inkjet system and to obtain detailed insight into the frequency and causes of jetting failures. Of all the substrates investigated, 96.2% were produced without any system or jetting failures. In 1.6% of the substrates, droplet emission failed and was correctly identified. Appropriate measures could then be taken to get the process back on track. In 2.2%, the imaging systems failed while droplet emission occurred correctly. In 0.1% of the substrates, droplet emission failure that was not timely detected occurred. Thus, the overall yield of the microarray manufacturing process was 99.9%, which is highly acceptable for prototyping.

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.

Fluid dynamical analysis of the distribution of ink jet printed biomolecules in microarray substrates for genotyping applications.

Oligonucleotide microarrays are tools used to analyze samples for the presence of specific DNA sequences. In the system as presented here, specific DNA sequences are first amplified by a polymerase chain reaction (PCR) during which process they are labeled with fluorophores. The amplicons are subsequently hybridized onto an oligonucleotide microarray, which in our case is a porous nylon membrane with microscopic spots. Each spot on the membrane contains oligonucleotides with a sequence complementary to part of one specific target sequence. The solution containing the amplicons flows by external agitation many times up and down through the porous substrate, thereby reducing the time delaying effect of diffusion. By excitation of the fluorophores the emitted pattern of fluorophores can be detected by a charge-coupled device camera. The recorded pattern is a characteristic of the composition of the sample. The oligonucleotide capture probes have been deposited on the substrate by using noncontact piezo ink jet printing, which is the focus of our study. The objective of this study is to understand the mechanisms that determine the distribution of the ink jet printed capture probes inside the membrane. The membrane is a porous medium: the droplets placed on the membrane penetrate in the microstructure of it. The three-dimensional (3D) distribution of the capture probes inside the membrane determines the distribution of the hybridized fluorescent PCR products inside the membrane and thus the emission of light when exposed to the light source. As the 3D distribution of the capture probes inside the membrane eventually determines the detection efficiency, this parameter can be controlled for optimization of the sensitivity of the assay. The main issues addressed here are how are the capture probes distributed inside the membrane and how does this distribution depend on the printing parameters. We will use two model systems to study the influences of different parameters: a single nozzle print head jetting large droplets at a low frequency and a multinozzle print head emitting small droplets at a high frequency. In particular, we have investigated the effects when we change from usage of the first system to the second system. Furthermore, we will go into detail how we can obtain smaller spot sizes in order to increase the spot density without having overlapping spots, leading eventually to lower manufacturing costs of microarrays. By controlling the main print parameters influencing the 3D distribution inside the porous medium, the overall batch-to-batch variations can possibly be reduced.