key: cord-0009994-3v7se14x
authors: Li, Cai‐Xia; Pan, Qian; Guo, Yong‐Gang; Li, Yan; Gao, Hua‐Fang; Zhang, Di; Hu, Hao; Xing, Wan‐Li; Mitchelson, Keith; Xia, Kun; Dai, Pu; Cheng, Jing
title: Construction of a multiplex allele‐specific PCR‐based universal array (ASPUA) and its application to hearing loss screening
date: 2007-12-27
journal: Hum Mutat
DOI: 10.1002/humu.20622
sha: 6bb22fc43e051f0384af85cd796533229ffdd108
doc_id: 9994
cord_uid: 3v7se14x

We demonstrate a new method, using a universal array approach termed multiplex allele‐specific PCR‐based universal array (ASPUA), and applied it to the mutation detection of hereditary hearing loss. Mutations in many different genes may be the cause of hereditary hearing loss, a sensory defect disorder. Effective methods for genetic diagnosis are clearly needed to provide clinical management. Owing to the broad genetic basis of this condition, clinical assay of such a highly heterogeneous disorder is expensive and time consuming. In ASPUA, the allele discrimination reaction is carried out in solution by multiplex allele‐specific PCR and a universal solid phase array with different tag probes is used to display the PCR result. The purpose of developing the ASPUA platform was to utilize the rapidity and simplicity of the amplification refractory mutation system (ARMS) with the detection power of microarray hybridization. This is the first report of the combination of these two technologies, which allow for the completion of allele‐specific detection of 11 of the most frequent mutations causing hereditary hearing loss in under 5 hr. The ASPUA platform was validated by accurately analyzing 141 patient samples that had been previously genotyped for GJB2, GJB3, SLC26A4, and MTRNR1. In addition, we also developed a simplified assay by using streptavidin‐coated magnetic beads instead of fluorescence for signal display that can be assessed through a conventional light microscope. We demonstrate that the ASPUA platform is rapid, cost‐effective, and easily‐used, and is especially appropriate for mutation detection in clinical genetic diagnostics. Hum Mutat 29(2), 306–314, 2008. © 2007 Wiley‐Liss, Inc.

Congenital hearing loss affects one in 1,000 live births and approximately 50% of these cases are hereditary [American College of Medical Genetics (ACMG), 2002; Morton, 1991] . Sequence variants of many genes can contribute to hereditary hearing loss (Hereditary Hearing loss Homepage; http://webhost. ua.ac.be/hhh). Connexin 26 gene (GJB2; MIM] 121011) molecular defects account for up to 50% of recessive nonsyndromic deafness with c.35delG, c.167delT, and c.235delC being the predominant GJB2 mutations in the Caucasian, the Ashkenazi Jewish, and the Japanese populations, respectively [Abe et al., 2000; Estivill et al., 1998; Rabionet et al., 2000] (Connexins and Deafness Homepage, now maintained by the Deafness Research Group [CRG] ; http://davinci.crg.es/deafness). Pendred syndrome (PDS) gene mutations in SLC26A4 (MIM] 605646) account for as much as 10% of hereditary deafness in diverse populations. The p.Leu236Pro (c.707T4C) and p.His723Arg (c.2168A4G) mutations account for 16% and 53% of all mutant alleles in Caucasoid and Japanese populations, respectively [Campbell et al., 2001; Friedman and Griffith, 2003; Tsukamoto et al., 2003 ]. In addition, persons with mtDNA MTRNR1 gene (MIM] 561000) mutation m.1555A4G are at risk of developing hearing loss if exposed to aminoglycoside antibiotics [Prezant et al., 1993] . Despite the large number of known rare mutations in many genes that may contribute to hearing loss, the patterns of high frequency mutations in populations and their population-wide distributions make it meaningful to initially select hotspot mutations for neonatal screening or genetic testing.

Various techniques are currently used to screen mutations, including restriction fragment length polymorphism (RFLP) [Hurd et al., 2002] , amplification refractory mutation system (ARMS) [Newton et al., 1989] , denaturing high-performance liquid chromatography (DHPLC) [Lin et al., 2001] , sequence analysis [Wang et al., 2002] , real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay [Bai and Wong, 2004] , and pyrosequencing [Ferraris et al., 2002] . However, most of these methods are low-throughput and time-consuming for multiple gene-based detection. Direct sequencing is the ''gold standard'' for assay of sequence variations, but is expensive and labor intensive.

In the recent few years microarray technology has had many applications in the areas of disease diagnosis and drug discovery. For example, we have developed protein microarrays for small molecule screening and severe acute respiratory syndrome (SARS) patient sample analysis [Du et al., 2005; Kong et al., 2005] , and also DNA microarrays for the early detection of SARS coronavirus as well as for the potential side effect study in developing siRNAbased hepatitis B virus (HBV) inhibiting drugs Guo et al., 2005] . DNA microarrays permit high-throughput mutation detection approaches, such as allele-specific primer extension on microarrays [Pastinen et al., 2000] , PCR/ligase detection reaction (LDR) universal array [Gerry et al., 1999] , microsphere-based single-base chain extension (SBCE) [Chen et al., 2000] , sequence-tagged molecular inversion probes (MIP) [Hardenbol et al., 2003] , and combinatorial sequencing-byhybridization (cSBH) [Cowie et al., 2004] . For the examination of hereditary hearing loss, there are several reports addressing the use of DNA microarray for the high-throughput screening of related DNA mutations [Cremers et al., 2007; Gardner et al., 2006; Siemering et al., 2006] . Although arrayed primer extension array (APEX) has recently been used to detect 198 sequence variations in deafness genes [Gardner et al., 2006] it is relatively expensive. There is great need for still simpler, faster, and more affordable approaches for routine genetic diagnostic laboratories. The purpose of developing ASPUA was to utilize the rapidity and simplicity of ARMS with the detection power of microarray hybridization to provide the allele-specific detection in under 5 hr. Here we used ASPUA to simultaneously screen 11 mutations causing hereditary hearing loss (Supplementary Table S1 ; available online at http://www.interscience.wiley.com/jpages/1059-7794/ suppmat). We also employed streptavidin-coated magnetic beads instead of fluorescence for labeling, to facilitate assay with the naked eye.

Various previously genotyped patient DNA samples were provided by the Chinese PLA General Hospital; the National Laboratory of Medical Genetics of China; Thomas Jefferson University; Stanford University School of Medicine, and Charles University, Prague. Genomic DNA was isolated from whole blood and plasmid DNA was extracted from E. coli strains with the Wizard s Genomic DNA Purification Kit and Wizard s Plus SV Minipreps DNA Purification System (Promega, Madison, WI), respectively. The extraction of genomic DNA from whole blood takes less than 1 hr. The QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) was used to introduce specific mutations into cloned genes at the 11 loci. Each gene insert was sequenced to verify that selected clones contained the desired mutations. The GenomiPhi V2 DNA Amplification Kit (GE Healthcare, Piscataway, NJ) was used for whole genome amplification of the genomic DNA (10 ng and 1 ng).The whole genome amplification product was purified by the NucleoSpin s Extract II kit (Macherey-Nagel, Düren, Germany).

All the primers and probes were synthesized and purified by Invitrogen (Invitrogen, Shanghai, China). The common primer and the universal primer were fluorescently labeled. Tags were designed and modified according to Chen et al. [2000] . The 5 0 -amino-tag probes with 15-nt poly T-linker adjacent to the 5 0end were covalently attached to aldehyde-modified glass slides (CapitalBio, Beijing, China) to capture the allele-specific PCR products. Each probe was resuspended at a concentration of 15 mM in 50% dimethyl sulfoxide (DMSO) and printed as five replica spots by SmartArrayer TM -48 Microarray Spotter (CapitalBio). The spot diameter was approximately 150 mm at 300 mm center-tocenter spacing.

Multiplex allele-specific PCR was carried out in two tubes to avoid interactions between some primer pairs. Four loci, c.35delG, c.547G4A, c.2168A4G, and c.919-2A4G (IVS7-2A4G) were amplified simultaneously in one tube, and the remaining seven loci were amplified in another tube. Reaction volumes were 15 ml, and contained 0.2 mM dNTPs, 2 mM MgCl 2 , 0.8 unit of HotStarTaq DNA Polymerase (Qiagen, Hilden, Germany), and 50 ng of genomic DNA or 5 pg of plasmid DNA. Optimal concentrations were determined separately for each primer. Asymmetric PCR was used to obtain sufficient single-strand DNA for hybridization. The amplification was performed in a PTC-225 Thermal Cycler (MJ Research, Watertown, MA). PCR conditions were as follows: 951C for 15 min; then: 941C for 30 sec, ramp 68 sec to 551C, hold for 30 sec; ramp 50 sec to 701C, hold for 45 sec for 10 cycles; then: 901C for 30 sec, ramp 60 sec to 551C, hold for 30 sec, ramp 50 sec to 701C, hold for 45 sec for 20 cycles; followed by 601C for 10 min, and 41C soak. Amplifications were thus completed within 2.4 hr. A negative PCR control (no template) was included for each run. No additional positive PCR control was needed because primers for both alleles are present in each reaction, and even for homozygous templates the product of at least one allele should always appear.

The products of both amplification reactions were combined. An aliquot of the mixture (5 ml) was suspended in 10 ml hybridization buffer (6 Â SSC, 5 Â Denhardt's reagent, 25% (v/v) formamide, 0.1% (w/v) SDS, 5 nM c-PC). After heating for 2 min at 981C and chilling on ice, the hybridization mixture was added to the subarray. The slide was incubated at 501C for 1 hr and washed twice (2 min each) at 421C in 0.3 Â SSC/0.1% SDS and in 0.06 Â SSC. Finally, the slide was centrifuged to dry.

The dried slide was imaged with a ScanArray s Express Microarray Scanner (PerkinElmer Life Sciences, Boston, MA). Laser power and photomultiplier tube (PMT) power were 90% and 70%, respectively. The signal intensities of the spots were quantified by GenePix Pro 4.0 (Axon Instruments, Foster City, CA). The absolute median signal intensity (AMSI) of each spot was calculated by subtracting the local background from the median signal intensity of the spot, and a minimum value of 1,000 was used as the cutoff. To exclude false-positive signals generated by primer dimers, AMSI for one allele was required to be at least 10 times the negative PCR control AMSI for that allele. If these criteria were met, the signal of one allele was considered to be positive.

The 5 0 -fluorescent dyes of the relevant oligonucleotides were replaced by biotin. The PCR, hybridization, and washing steps were conducted as described above. Streptavidin-coated MyOne TM Dynalbeads (Dynal Invitrogen, Oslo, Norway) were pretreated according the protocol from the supplier, and then 20% goat blood serum in 1 Â binding & washing buffer (5 mM Tris-HCl (pH7.5), 500 nM EDTA, 1 M NaCl) was added to reduce nonspecific background adhesion. The solution was added into the array block. After incubation for 10 min for streptavidin-biotin binding, the array was washed twice (2 min each) in 2 Â SSC/0.1% SDS and in 0.2 Â SSC. Finally, the image was viewed with Leica DMRE microscope (Leica Microsystems, Wetzlar, Germany) and captured by Canon EOS-1Ds Mark II digital camera EF100 mm f/2.8 Macro USM (Canon, Tokyo, Japan).

An outline of multiplex ASPUA is given in Figure 1 . Multiplex allele-specific PCR is carried out using HotStarTaq DNA Polymerase, and then the PCR products are used to hybridize with capture probes on a universal array. Results are determined by the signal intensity and by the position of the tag probe bound on the array. For each locus, two tagged allele-specific primers that differ at their 3 0 -terminal base define the allele. The HotStarTaq DNA Polymerase used lacks a 3 0 to 5 0 exonuclease activity, thus if a PCR primer has a mismatch with the template at its 3 0 -terminal base, the efficiency of amplification will be greatly reduced. The common primer is tailed, and a universal primer that has the same sequence as the tail is also used to increase the amount of the single-strand DNA . In the ASPUA assay, the universal array serves as a decoding tool to display the results of the previous amplification. We first validated the universal array to ensure the specificity of the hybridization results.

Speci¢city of ASPUA Figure 2A shows the array format, in which QC and BC were the positive and negative controls of spotting efficiency, and PC and NC were oligonucleotides that served as positive and negative controls of hybridization, respectively. PC was complementary to a TAMRA-labeled oligonucleotide in the hybridization mixture, while NC was noncomplementary to any sequence in the mixture. The rest of the spots on the subarray were tag probes that captured the ASPUA PCR products.

To find out if the allele-specific primers can only amplify their intended target allele and if different alleles of these mutations can be discriminated specifically, a homozygous DNA sample was amplified in the presence of mixtures of all primers. Only the intended target alleles were found to be amplified. For those mutations for which we could not obtain homozygous patient samples, plasmid DNA clones of single alleles were used instead. Genomic DNA samples were used as source templates for the mutations (c.35delG, c.167delT, c.176_191del16, c.235delC, c.299_300delAT, m.1555A4G, and c.919-2A4G) and for the wild-type, and for each type of sample all 11 loci should be detected. The remaining tests were on cloned single-allele templates in which only homozygous signals are detected. The p.Arg180X and p.Glu183Lys mutations occur closely within GJB3 gene (MIM] 603324), and were constructed in a nonoverlapping manner in one plasmid. Other mutations resided on their own plasmid individually. As shown in Figure 2B , only the tags related to the correct primers for the template showed a significant signal. Specific signals appeared for each mutant gene template. Table 1 3

Denaturing Hybridization C Tagn Tag1Tag2 FIGURE 1. The principles of ASPUA. A: Multiplex AS-PCR was conducted in two tubes (2.5 hr).The ampli¢cation was asymmetric and generated su⁄cient single-stranded labeled DNA for hybridization and detection. B: After denaturing, the PCR products were hybridized with the universal array (1hr). C: The washed array was read by an array scanner.The image was analyzed to obtain the genotyping results of multiple loci (0.5 hr).

shows the AMSI of the specificity study and the standard deviation (SD) data of five repeated experiments. Each of the positive signals in all five repeated experiments meets the two criteria listed in Materials and Methods.

The introduction of artificial mismatches near the 3 0 -end of the allele-specific primers (AS primers) could enhance the selective hybridization specificity [Lo et al., 1991; Newton et al., 1989] . Reducing the concentration of the AS primer can also increase the discrimination power of this method; however, the efficiency of the two measures varied over a range of PCR conditions. To study these effects we have chosen the wild-type primer of m.1555A4G mutation as the assay target as it has a T residue at the 3 0 -end and is often poorly discriminated (data not shown). Artificial mismatch was introduced to the AS primer at positions three, five, and nine bases from the 3 0 -end (called m.1555A4G-W3, -W5, and -W9), respectively. The discrimination ratio (DR) of the natural wild-type primer is low because of the T-G mismatch between the wild-type primer and the mutant target. Decreasing the primer concentration can increase the specificity, but this measure also greatly reduces the amplification efficiency. In contrast, the presence of artificial mismatches near the 3 0 -end of primers can enhance the discrimination ratio with little deleterious influence on the amplification efficiency. As the artificial mismatch is positioned further from the 3 0 -end the specificity is seen to decrease. The m.1555A4G-W3 primer maintains high discrimination specificity during signal amplification in the presence of increasing amounts of mutant template, or increasing concentrations of magnesium. In conclusion, the m.1555A4G-W3 primer appears to perform well over a wide range of PCR conditions.

We tested 141 normal and patient DNA samples that had been previously genotyped. The genotypes detected by ASPUA and the clinical information of these samples are summarized in Supplementary Table S2 . Among the 141 individuals examined, 122 samples were from deaf patients and nine were from carriers. One additional patient was found to be homoplasmic for the m.1555A4G mutation but displayed no clinical manifestations, and no mutations were detected in the nine remaining control samples from normal individuals. For each mutation, the AMSI value of the two possible alleles was plotted on each axis. An individual data point falls into one of the three clusters and is assigned the genotype of AA, AB, or BB. All of the expected 1,551 calls were generated for the 141 samples. To evaluate the minimum amount of sample required for ASPUA, we assayed various amounts of a wild-type gDNA ( Supplementary Fig. S1 ). The AMSI of the sensitivity study and the SD data of five repeated experiments is shown in Table 2 . Attenuation of AMSI was observed with decreased amount of target. AMSI values higher than 1,000 were observed at all loci at the 25-ng genomic template, but at 10 ng signals of four of the loci fell below 1,000. We explored using multiple displacement amplification (MDA) to expand the detection sensitivity of ASPUA. Supplementary Figure S1 shows that both 10 ng and 1 ng of starting gDNA could be detected by ASPUA after amplification by MDA, whereas a MDA control reaction with no template was blank and the no target control MDA product showed no significant signals.

To achieve visible light-assisted detection, streptavidin-coated MyOne TM Dynalbeads were used to display the hybridization results of the 11 mutations (Fig. 3) . The streptavidin-coated beads are 1.05 mm in diameter. They can bind covalently to the biotinlabeled PCR product on the microarray. Those beads that are not specifically bound will be removed by conventional washing. The beads remaining bound on the array can be viewed with an optical microscope or by naked eye. Previously genotyped samples were used for this study. The array format is the same as that shown in Figure 2A . From Figure 3 , it is clear that all the samples were detected specifically. Three repeated experiments were conducted to confirm the accuracy of the detection results.

ASPCR, also known as ARMS [Newton et al., 1989] , is an established method for genotyping mutations. In ARMS, the PCR product is separated by gel electrophoresis. The protocol usually detects one allele per reaction and is laborious when testing many mutations in different samples. To overcome this problem, Roberts et al. [2000] developed multiplex ARMS, in which two separate PCR reactions (ARMS1 and ARMS2) containing a mixture of wild-type or mutation specific primers are performed for each sample. The genotyping was determined by the size of PCR products on the gel image. Gómez-Llorente et al. [2004] combined allele-specific PCR with capillary electrophoresis. This strategy needs different fluorescent dyes and specialized separation equipment, which increases the cost and limits the flexibility of the assay.

Here, we have combined a multiplex allele-specific PCR with a universal array (ASPUA) for rapid and cost effective simultaneous genotyping of 11 mutations causing hereditary hearing loss. The mutations, ranging in size from single-base changes to large 16-bp deletions could be accurately identified. Homozygous DNA samples were used to assess the specificity of the entire assay. The specificity of both the enzymatic reaction and the universal array hybridization conferred the high discrimination power to the entire procedure. Even mutations that are adjacent, such as the two loci nine bases apart in the GJB3 gene [Xia et al., 1998 ], can be accurately detected.

During optimization of the assay we observed that the use of HotStarTaq DNA Polymerase was crucial to performance of multiplex ASPUA, as it can prevent the formation of primer dimers to a large extent. Primer dimers both reduce the efficiency of amplification of some alleles, and cause some false-positive signals to be generated and detected at the PCR negative control. A minimum 25 ng of gDNA is enough for efficient ASPUA detection in the 15-ml reaction volume. If the amount of patient sample is limiting, MDA can be used to increase template abundance before ASPUA allowing as little as 1 ng of starting gDNA to be successfully genotyped. Artificial mismatches can also be introduced close to the 3 0 -end to enhance the specificity of AS primers by lowering the stability of the primer/template complex, preventing the mismatched primer from extending efficiently. Artificial mismatches can improve the specificity greatly and discriminate efficiently over a wide range of PCR conditions.

In the patient study, we tested 141 samples to validate the accuracy of ASPUA, of which 18 of the samples were blind test samples. The results of 141 samples were fully concordant with the initial genotype information of the supplier laboratories. Signal intensity variation was found among different samples. Because the patient DNA samples were supplied by several laboratories, the DNA quality varied to some degree. But this difference did not influence the assay outcomes and all the positive signals meet the assay criteria. One sample was found to carry the m.1555A4G mutation but the patient showed no clinical manifestations. For such a patient, aminoglycoside antibiotics should be avoided to prevent induced development of deafness. During preparation of this manuscript, we applied ASPUA to a further 515 patient samples and the results were fully concordant with direct sequencing.

We have validated the ASPUA platform with only 11 mutations, many less than reported using APEX methods [Gardner et al., 2006; Cremers et al., 2007] . The low number of abundant mutations detected by the current ASPUA microarray platform was intentional, as it allowed for sufficient numbers of samples of each genotype to be collected for regulatory-requested, statistically-verifiable data. In the future, we need to analyze more samples to ensure the accuracy of the ASPUA platform over a wider diversity of quality of patient samples, and over a wider range of different genotype combinations. Most of the mutations in this work are frequently found in Chinese hearing loss patients. For example, our preliminary study on 1,190 Chinese nonsyndromic hearing impairment (NSHI) patients found that the c.235delC mutation accounts for 88.8% (222/250) of the patients who carry the GJB2 mutation and the c.299_300delAT and c.176_191del16 mutation was found in 24.8% (62/250) and 7.6% (19/250) of these patients, respectively, as some of the patients carry compound mutations. Dai et al. [2006] studied 38 Chinese patients with enlarged vestibular aqueduct (EVA) and found that 91.4% of the patients carry at least one mutation in the SLC26A4 gene, and patients who carry the c.919-2A4G (IVS7-2A4G) and p.His723Arg mutation accounted for 71.9% and 25%, respectively. Liu et al. [2006] reported 3.43% (63/1,836) of the Chinese NSHI patients carry the m.1555A4G mutation. The Connexin 31 gene (GJB3) was first cloned in China, and the p.Arg180X and p.Glu183Lys mutations were associated with hearing loss [Xia et al., 1998 ]. Three mutations that are prevalent in Caucasoid or Ashkenazi Jewish patients were also chosen to expand the detection spectrum of our methods. For example, the c.35delG mutation accounted for 85% of GJB2 mutations in families with recessive nonsyndromic deafness from Spain and Italy [Estivill et al., 1998 ] and the c.167delT mutation accounted for 40% of the mutant alleles in the Ashkenazi Jewish population [Sobe et al., 2000] . The p.Leu236Pro mutation accounts for 16% of all SLC26A4-disease causing alleles in the Caucasoid population [Campbell et al., 2001] .

We intend to develop a second generation of hereditary deafness microarray by increasing the range of mutations examined by the ASPUA assay. We have shown that separation of amplification reactions into different tubes with compatible combinations of primers and HotStar DNA polymerase permits efficient amplification, even with a range of DNA qualities and quantities. Increasing the number of reaction tubes to provide for additional alleles and gene loci is the simplest approach to increase gene testing, and the addition of a few new gene loci to each tube could also be readily tested, and if efficiency remains high, adopted. We have also demonstrated that alleles with low hybridization efficiency can be markedly improved by inclusion of deliberate mismatches near to the 3 0 -discriminatory nucleotide-a measure that could be tested with any newly added suboptimal alleles.

The ASPUA platform is simple to use, and although it is currently low-throughput, it can readily be expanded. Other deafness analysis platforms have different design formats. The allele-specific oligonucleotide biochip of Siemering et al. [2006] involves seven steps from PCR to interpretation of results and takes more than 1 day to perform. The APEX method [Gardner et al., 2006] involves six steps and is completed in 6 hr, yet it employs multichannel scanners and four-color fluorescence labeling, which adds considerable expense to the setup and running costs. In contrast, current ASPUA analysis of four samples on a four-subarray chip can be completed in 5 hr with only four steps: DNA extraction (1 hr), PCR (2.5 hr), hybridization (1 hr), scanning and automatic characterization of results (0.5 hr), avoiding the need for amplicon purification and several other steps required by other platforms. The genetic heterogeneity of deafness makes it necessary to undertake multigene detection. Yet, with the current method one could readily increase either the number of genes or the number of mutations to about 50 with almost no increase in the time for analysis. The number of samples analyzed could also be increased with some small time cost. If say 48 samples were analyzed (requiring 12 four-subarray chips) the total analysis time, including scanning and interpretation, would increase by about a further 40 minutes due to sample handling.

Although DNA sequencing may generate fully analyzed genotype data as rapidly as ASPUA, the cost of capital equipment required for sequencing may be several times that of our technology. However, conventional methods such as RFLP or dot blot allele specific oligonucleotide (ASO) both become labor-intensive when the number of mutations or samples is increased. In ASPUA, the PCR products can be used to hybridize directly without the need of post-PCR purification or dye-labeled ddNTP extension, which saves some costs. The flexibility of the universal array also contributes to the cost reduction because the same universal array format can be developed for use for completely different gene mutation assays. It is desirable to avoid the use of expensive instruments and to limit costs significantly for general clinical diagnostic settings and to provide a low cost method for laboratories in less developed regions of China. To this end we have employed magnetic bead-labeling to achieve visible light-assisted detection. The hybridization results can then be visualized through a light microscope or by naked eye. Several groups have also addressed the development of simple and specific methods with the minimal instrumentation requirements. Taton et al. [2000] developed a sandwich hybridization DNA array using oligonucleotide-modified gold nanoparticle probes coupled with signal amplification by use of silver deposition. The hybridization results could be visualized by a flatbed scanner. However, compared to the sandwich hybridization assay, ASPUA is more flexible and simple.

In summary, we have combined allele-specific PCR and universal array methodologies for the detection of mutations causing hereditary hearing loss. The specificity and simplicity of the ASPCR reaction provides high genotyping accuracy. ASPUA offers several distinct advantages for routine diagnostic laboratories. Here we have employed it for multiplex detection of 11 mutations causing hereditary hearing loss. The ASPUA universal array makes the multiplex detection highly flexible, and the numbers of gene loci detected can be increased if warranted. The microarray could also be redesigned with a greater number of subarrays so that much larger numbers of patient samples could be tested simultaneously. Finally, the demonstrated feasibility of visible light-assisted detection avoids the need for special detection equipment. All of these features should make it easily acceptable in clinical genetics laboratories.

Prevalent connexin 26 gene (GJB2) mutations in Japanese

Genetics evaluation guidelines for the etiologic diagnosis of congenital hearing loss. Genetic evaluation of congenital hearing loss expert panel. ACMG statement

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