Abbate, Rosanna

Bernabini, S.

Frusconi, S.

Giotti, I.

Giusti, Betti

Pepe, Guglielmina

Poggi, F.

Rossi, Luigi

Torricelli, Francesca

English

Florence Research

produce a peak within 1–2 °C of the predicted Tm. Thisallows Tm peaks, which indicate the presence or absenceof a PCR product, to be strategically placed within thetypical melting curve range of 45–70 °C for hybridizationprobes. Over this melting curve range we feel it should bepossible to clearly distinguish three peaks in a singlechannel. This potentially makes it possible to multiplexsix mutations in two capillaries when both fluorescentchannels are used. This enhances the capabilities of theLightCycler for mutation detection; similar strategiescould also be used for other real-time systems.The results presented here demonstrate the ability tocarry out multiplex mutation detection by use of a com-bination of ARMS PCR and real-time detection. A labora-tory currently using ARMS PCR in a diagnostic settingcan quite easily convert the standard ARMS PCR to areal-time ARMS PCR. This could have a major advantagein time savings and reduce the handling of potentialcarcinogenic ethidium bromide.References1. Collins F. Cystic fibrosis: molecular biology and therapeutic implications.Science 1992;256:774–9.2. Tsui LC. Cystic fibrosis mutation database. http://www.genet.sickkids.on.ca (Accessed November 2003).3. Scotet V, Barton DE, Watson JB, Audrezet MP, McDevitt T, McQuaid S, etal. Comparison of the CFTR mutation spectrum in three cohorts ofpatients of Celtic origin from Brittany (France) and Ireland. Hum Mutat2003;22:105.4. Ravik-Glavac M, Glavac D, Dean D. Sensitivity of single-stranded conforma-tion polymorphism and heteroduplex method for mutation detection in thecystic fibrosis gene. Hum Mol Genet 1994;3:801–7.5. Ferrie RM, Schwarz MJ, Robertson NH, Vaudin S, Super M, Malone G, et al.Development, multiplexing and application of ARMS tests for commonmutations in the CFTR gene. Am J Hum Genet 1992;51:251–62.6. Kleinle S, Tabiti K, Gallati S. Limitations of melting curve analysis using SybrGreen 1—fragment differentiation and mutation detection in the CFTR-gene.In: Dietmaier W, Wittwer C, Sivasubramanian N, eds. Rapid cycle real-timePCR methods and applications (genetics and oncology). Berlin: Springer,2002:47–56.7. Burggraf S, Malik N, Schumacher E, Olgemo¨ller B. Rapid screening for fivemajor cystic fibrosis mutations by melting peak analysis using fluorogenichybridisation probes. In: Dietmaier W, Wittwer C, Sivasubramanian N, eds.Rapid cycle real-time PCR methods and applications (genetics and oncol-ogy). Berlin: Springer, 2002:85–94.8. Glaab W, Skopek TR. A novel assay for allelic discrimination that combinesthe fluorogenic 5	 nuclease polymerase chain reaction (TaqMan®) andmismatch amplification mutation assay. Mutat Res 1999;430:1–12.9. Schu¨tz E, Von Ahsen N. Spreadsheet software for thermodynamic meltingpoint prediction of oligonucleotide hybridization with and without mis-matches. Biotechniques 1999;27:1218–22.10. Bernard PS, Ajioka RS, Kushner JP, Wittwer CT. Homogeneous multiplexgenotyping of hemochromatosis mutations with fluorescent hybridizationprobes. Am J Pathol 1998;153:1055–61.11. Von Ahsen N, Oellerich M, Armstrong VW, Schu¨tz E. Application of athermodynamic nearest-neighbor model to estimate nucleic acid stabilityand optimize probe design: prediction of melting points of multiplemutations of apolipoprotein B-3500 and factor V with a hybridizationprobe genotyping assay on the LightCycler. Clin Chem 1999;45:2094–101.DOI: 10.1373/clinchem.2003.030445Improvement of Low-Density Microelectronic ArrayTechnology to Characterize 14 Mutations/Single-Nucleotide Polymorphisms from Several Human Geneson a Large Scale, Sabrina Frusconi,1†* Betti Giusti,2† LucianaRossi,2 Sara Bernabini,1 Filippo Poggi,2 Irene Giotti,1 RosannaAbbate,2 Guglielmina Pepe,2 and Francesca Torricelli1(1 Unita’ Operativa Citogenetica e Genetica, Azienda Os-pedaliera Careggi, Florence, Italy; 2 Dipartimento AreaCritica Medico Chirurgica, University of Florence, Flo-rence, Italy; † these authors contributed equally to thiswork; * address correspondence to this author at: Unita’Operativa Citogenetica e Genetica, Azienda OspedalieraCareggi, Viale Morgagni 85, 50134 Florence, Italy; fax39-055-4279686, e-mail genomica@ao-careggi.toscana.it)Large-scale human genetic studies require new technolo-gies to genotype several samples with relative ease, highaccuracy, and reasonable costs. Among the availableapproaches, a microelectronic array technology has beendeveloped for DNA hybridization analysis of mutations/single-nucleotide polymorphisms (SNPs) (1–4). The mi-croelectronic array system (NanoChip® Molecular Biol-ogy Workstation; Nanogen) produces a defined electricfield that allows charged molecules, such as nucleic acids,to be transported to any test site, or pad, on the electronicchip (NanoChip cartridge). Electronic-based molecule ad-dressing can rapidly achieve a high concentration ofamplicons on each pad of the cartridge. Control of tem-perature allows use of an optimal thermal stringency tocharacterize a SNP/mutation in all 100 pads of a cartridgesimultaneously (5, 6). A thin hydrogel permeation layeroverlies the pads; the presence of avidin or streptavidinin this layer allows the binding of biotinylated PCRproducts.Although the technology is attractive, only a few pro-tocols for its use have been published (7–11). We describethe development, optimization, and validation of a high-throughput method for SNPs and mutations analysis thatallows performance of 1372 characterizations on eachchip.We studied samples from 150 individuals for 14 SNPs/mutations previously characterized by standard methods(restriction analysis, automatic sequencing, and allelicdiscrimination). Genomic DNA was isolated from periph-eral blood by use of the FlexiGene DNA reagent set(QIAGEN GmbH). We analyzed 14 DNA mutations/SNPs for a total of 2100 characterizations (homozygouswild type, n  1367; heterozygous, n  522; homozygousmutant, n  211). The 14 nucleotide substitutions wereSNPs/mutations involving the glycoprotein Ia (GpIa),glycoprotein IIIa (GpIIIa), follicle-stimulating hormonereceptor (FSHR), hereditary hemochromatosis (HFE), and1 chain of collagen type 6 (COL6A1) genes.For each mutation/SNP we designed a set of probesconsisting of a forward and a reverse PCR oligonucleotide(one oligonucleotide for each SNP/mutation was 5	-biotinylated); two reporter oligonucleotides (one labeledwith Cy3, specific for the wild-type nucleotide; the other,labeled with Cy5, specific for the mutant nucleotide), andClinical Chemistry 50, No. 4, 2004 775one stabilizer oligonucleotide. Reporter oligonucleotideshad Cy3 or Cy5 fluorophore at the 5	 end and a SNP/mutation at the 3	 end. The stabilizer was located imme-diately downstream from the 3	 end of reporter oligonu-cleotides. All of the designed oligonucleotides are listed inTable A of the Data Supplement that accompanies theonline version of this Technical Brief at http://www.clinchem.org/content/vol50/issue4/.To genotype the 14 SNPs/mutations, we amplifiedseven DNA sequences by PCR. The seven amplicons werepooled and desalted with the Nucleo Fast system (96-wellPCR plates) from Macherey-Nagel. We mixed 30 L ofeach 55-L desalted amplicon pool with 30 L of 100mmol/L histidine. Four wells containing solutions of 0.3mol/L NaOH, 0.1 mol/L NaOH, and 50 mmol/L histi-dine and a known heterozygous sample for each SNP/mutation analyzed were also included on the plate. Theloader was programmed to electronically address eachamplicon pool to specific pads on the cartridge. The 50mmol/L histidine solution without amplicons was ad-dressed to two pads in each cartridge as background.Each pool of samples was analyzed on one pad in twodifferent cartridges, whereas two known heterozygoussamples for each mutation/SNP were addressed to fourpads in each cartridge. Each sample was amplified in asecond independent PCR reaction, pooled, and addressedto one pad of a third cartridge. A second amplificationreaction was also carried out for the two known heterozy-gous samples for each mutation/SNP, which were pooledand addressed to four pads of the third cartridge.One set of stabilizer and reporter oligonucleotides at atime, specific for one SNP/mutation, was hybridized tothe cartridge. The hybridization mixture was then re-moved, and the chip was rinsed twice with high-saltbuffer. The cartridge was then placed in the reader, wherethe temperature was increased to the optimum tempera-ture for discrimination (see Table A of the online DataSupplement). After each hybridization and scanning pro-cedure, a stripping step was performed to remove report-ers and to allow hybridization with another set of probes.The same protocol was applied to analyze each mutation/SNP. To study all 14 SNPs/mutations, we performed 14hybridization/stripping steps. A fluorescence scan wasperformed with the system reader. The software directlyassigned the genotype to each sample (Table 1). Knownheterozygous control samples were used as Cy3:Cy5fluorophore ratio references for signal normalization. Forbackground subtraction, fluorescence signals in the histi-dine pads were used. The mean values for Cy3 and Cy5fluorescence were determined by averaging the amountof fluorescence in each pad. The Cy5:Cy3 ratio for eachpad in the heterozygous control set was averaged to-gether to build the scale. The scaled mean was determinedby multiplying the scale and the mean. The ratios of thescaled means were used to make the genotype designa-tion. For this purpose the Nanogen-recommended criteriawere used: heterozygous if the Cy5:Cy3 ratio was be-tween 3:1 and 1:3; homozygous for the wild-type andmutant alleles if the ratios were 1:5 and 5:1, respec-tively. Ratios between 1:3 and 1:5 for the wild-type orbetween 3:1 and 5:1 for the mutant alleles indicated a needto repeat the analysis. For pad exclusion, the signal-to-noise ratio was set to a default at 5:1.Fluorescence intensities were similar for the same PCRproducts addressed to different pads of the same car-tridge and in different pads of a different cartridge. Majordifferences in signal intensities were observed in the caseof the second amplification addressed to a different car-tridge. After the normalization procedure, all heterozy-gous samples showed ratios very close to 1, ranging from1.0 to 1.53 [mean (SD), 1.11 (0.13)] in adherence with datarelative to the known heterozygous samples shown inTable B of the online Data Supplement.No background increase was detected after the 14thhybridization. The background signal ranged from 1.5 to15 independent of the SNP/mutation analyzed and wasnot related to the number of hybridization/strippingsteps performed.We found 100% concordance between the results ob-tained by the approach described here and those obtainedby standard techniques (automated sequencing, allelicdiscrimination, and restriction analysis) either within orbetween assays. This concordance was observed for all ofthe samples independent of the genotype (homozygouswild type, 1367 of 1367; heterozygous, 522 of 522; ho-mozygous mutant, 211 of 211), the SNPs/mutations stud-Table 1. Example of all data generated by the analysis software for a heterozygous, a homozygous wild-type, and ahomozygous mutant sample.aa R, red (Cy5 fluorescence); G, green (Cy3 fluorescence); SNR, signal-to-noise ratio.776 Technical Briefsied, and the comparison method. In 7% of the samples thesignal-to-noise ratio was low. The software failed toassign a genotype because of a PCR failure in 5% ofsamples and because of an insufficient amount of PCRproduct in 2% of samples.Our data are in agreement with other reports on theNanogen technology (7, 9, 10), but our approach reducedthe time for the detection of a SNP/mutation from 5 min(manufacturer’s protocol) to 0.7 min. The manufactur-er’s protocol required 8 h for loading of 98 PCR products,one hybridization step, one washing step, and 98 detec-tions. The longest time (7 h) was required to address the98 samples to the pads. Our protocol took 7 h for theaddressing of 98 samples, each containing 7 amplicons,and 8 h to perform 14 hybridizations, 14 washing steps,and 14  98 detections. An additional factor to be consid-ered is the cost per assay: reagents in the protocol sug-gested by the manufacturer cost approximately €9 foreach detection (DNA extraction, PCR, purification, chip,and probes). The simultaneous analysis of multipleSNPs/mutations on each pad provided by our protocolreduces the price to approximately €2 for each SNP/mutation. This reagent cost is similar to or less expensivethan the reagent costs for the standard techniques (allelicdiscrimination, restriction analysis, and sequencing), withreagent cost ranges of €2–25. In addition, the shorteranalysis time for our protocol reduces technician time andprovides increased numbers of results over which toamortize the cost of the instrument. Finally, our protocolallows optimization of procedures in only 1 h for eachSNP/mutation.To the best of our knowledge, this is the first reportdescribing the multiple analysis of 14 SNPs/mutationsinvolving 5 human genes (7 amplicons) on each pad of thesame microelectronic chip without affecting the accuracyof the analysis.The easy preparation and analysis of samples, the lowcost per sample, the reduction in the time required for theanalysis, and the concordance of the results with thecomparison methods demonstrate that this approach im-proves the throughput of microelectronic chip technol-ogy. We believe that the Nanogen technology is a prom-ising tool for molecular diagnosis, pharmacogenetics, andresearch.We thank Dr. Bjo¨rn Hihn for assistance during this study.References1. Edman CF, Raymond DE, Wu DJ, Tu E, Sosnowski RG, Butler WF, et al.Electric field directed nucleic acid hybridization on microchips. Nucleic AcidsRes 1997;25:4907–14.2. Sosnowski RG, Tu E, Butler WF, O’Connell JP, Heller MJ. Rapid determinationof single base mismatch mutations in DNA hybrids by direct electric fieldcontrol. Proc Natl Acad Sci U S A 1997;94:1119–23.3. Gilles PN, Wu DJ, Foster CB, Dillon PJ, Chanock SJ. Single nucleotidepolymorphic discrimination by an electronic dot blot assay on semiconductormicrochips. Nat Biotechnol 1999;17:365–70.4. Radtkey R, Feng L, Muralhidar M, Duhon M, Canter D, DiPierro D, et al.Rapid, high fidelity analysis of simple sequence repeats on an electronicallyactive DNA microchip. Nucleic Acids Res 2000;28:e17.5. Umek RM, Lin SW, Vielmetter J, Terbrueggen RH, Irvine B, Yu CJ, et al.Electronic detection of nucleic acids. J Mol Diagn 2001;3:74–84.6. Heller JM, Forster AH, Tu E. Active microelectronic chip devices which utilizecontrolled electrophoretic fields for multiplex DNA hybridization and othergenomic applications. Electrophoresis 2000;21:157–64.7. Santacroce R, Ratti A, Caroli F, Foglieni B, Ferraris A, Cremonesi L, et al.Analysis of clinically relevant single-nucleotide polymorphisms by use ofmicroelectronic array technology. Clin Chem 2002;48:2124–30.8. Sosnowski R, Heller MJ, Tu E, Forster AH, Radtkey R. Active microelectronicarray system for DNA hybridization, genotyping and pharmacogenomicapplications. Psychiatr Genet 2002;12:181–92.9. Evans JG, Lee-Tataseo C. Determination of the factor V Leiden single-nucleotide polymorphism in a commercial clinical laboratory by use ofNanoChip microelectronic array technology. Clin Chem 2002;48:1406–11.10. Erali M, Schmidt B, Lyon E, Wittwer C. Evaluation of electronic microarraysfor genotyping factor V, factor II, and MTHFR. Clin Chem 2003;49:732–9.11. Thistlethwaite WA, Moses LM, Hoffbuhr KC, Devaney JM, Hoffman EP. Rapidgenotyping of common MeCP2 mutations with an electronic DNA microchipusing serial differential hybridization. J Mol Diagn 2003;5:121–6.DOI: 10.1373/clinchem.2003.025197Clinical Chemistry 50, No. 4, 2004 777

Improvement of low-density microelectronic array technology to characterize 14 mutations/single-nucleotide polymorphisms from several human genes on a large scale.

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Improvement of low-density microelectronic array technology to characterize 14 mutations/single-nucleotide polymorphisms from several human genes on a large scale.

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