Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

Abstract

DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40 degrees C. When processed in the MTAW, probes selected without considering melting temperature resulted in improved genotyping compared with probes selected according to theoretical melting temperature and run under one condition. In conclusion, the MTAW is a versatile tool that can facilitate screening of a large number of probes for genotyping assays and can also enhance the performance of diagnostic arrays.

Figure 1.

The multi-thermal array washer (MTAW). (A) Photograph showing the experimental setup used to wash slides in the device. (B) Photograph of the assembled MTAW. (C) Schematic drawing of an assembled system viewed from the side. The main parts of the device are shown: (i) the PCB board, microheaters, and thermistors, including a slide holder attached to a layer of polydimethylsiloxane (PDMS); (ii) the PDMS layer, which defines the fluidic channels and chambers above the heaters; (iii) the microscope slide printed with identical subarrays that face towards corresponding chambers after the device is sealed.

Figure 2.

Scanning images of a processed array and corresponding melting curves. The scanning images show an array of T_m-matched probes (A) and three re-designed shorter probe-pairs (B) hybridized with amplified and labeled target material that was obtained from a person heterozygous for the CD8/9+G mutation and was processed in the MTAW. The temperatures in the respective zones are denoted to the left, and the identities of the probes are indicted below the images. Wild-type probes (Wt) for the different mutations and the corresponding mutant probes (Mt) are shown at the bottom and the top of each panel, respectively. (C) Melting curves of the wild-type (square) and mutant (diamond) probes at three different mutation sites. The graphs are based on the quantified signal intensity (in arbitrary units, a.u.) of the scanning images obtained at the corresponding temperatures. The secondary Y-axes present the normalized ratio (triangle; see Materials and Methods) calculated from the signals of the wild-type and mutant probes at each of the indicated temperatures.

Figure 3.

Genotyping of patient material using the MTAW and a T_m-matched probe set. Thirty-two different samples were individually hybridized to arrays of probes and subsequently processed in the MTAW. For each mutation site, a graph shows the normalized ratios (see Materials and Methods) at the indicated temperatures. Symbols: diamonds, the average value of all samples carrying the wild-type DNA sequence on both alleles (29 samples for all but CD8/9 and IVS I + 5, which have 27 and 30 respectively); error bars, the minimum and maximum observed ratios; dashes, the normalized ratios for heterozygous samples (three samples for each mutation site, except IVS I + 5 with only two); triangles, the normalized ratios for homozygous mutation in position CD8/9 (two samples).

Figure 4.

Genotyping of patient material using the MTAW and shorter redesigned probes. Thirty-two different samples were individually hybridized to arrays of shorter redesigned probes and subsequently processed in the MTAW. Calculation of normalized ratios is explained in the Materials and Methods section, and the symbols used are as described in the legend of Figure 3. The numbers within parentheses indicate the lengths of the probes (wild-type/mutant).

Figure 5.

Comparison of different methods used for genotyping in the MTAW. We define successful probe pair performance when homozygotes have normalized ratio of ≥0.7 (wild types) or ≤0.3 (mutants) and heterozygotes have normalized ratio between 0.35 and 0.65. The hatched lines in the graphs indicated these boundaries. (A) T_m-matched probe set in which all probes were washed at the single optimal temperature of 37°C. (B) T_m-matched probe set in which each probe pair was washed at its optimal temperature, so that the normalized ratio of the heterozygotes was as close to 0.5 as possible. (C). A mixed set of probe pairs, all of which originated from the T_m-matched probe set, except those towards the CD24 and CD27/28 positions, which were substituted with shorter probes. The ratio shown for each probe pair at the temperature (°C) that was optimal for clear classification of genotypes, which is indicated by the value given above each mutation. The probe sets used are indicated as follows: T_m stands for probes from the T_m-matched set; 13/13 denotes the 13-nt probes used for the CD24 mutation; 12/13 designates the 12-nt wild-type probes and the 13-nt mutant probes used to detect the CD27/CD28 mutation. Computation of the normalized ratio is explained in the Materials and Methods section, and the symbols used are described in the legend of Figure 3.