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1 Department of Pediatrics, The Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.
2 Unita’ di Ricerca in Aterosclerosi e Trombosi, IRCCS Casa Sollievo della Sofferenza, 71013 San Giovanni Rotondo, Italy.
3 Dipartimento di Science Neurologiche, Ospedale Maggiore Policlinico, University of Milan, 20122 Milan, Italy.
4 Laboratorio di Genetica Molecolare, Istituto G. Gaslini, 16148 Genova, Italy.
5 Unità di Genomica per la Diagnostica delle Patologie Umane, IRCCS H. San Raffaele, Diagnostica e Ricerca San Raffaele S.p.A., 20132 Milan, Italy.
6 Dipartimento di Medicina Sperimentale e Patologia, Università "La Sapienza", 00198 Roma; IRCCS–C.S.S. San Giovanni Rotondo and C.S.S.–Mendel, 00198 Rome, Italy.
7 Laboratorio di Biologia Molecolare, Ospedale Infantile Regina Margherita, 10124 Torino, Italy.
8 Department of Medicine, Cardeza Foundation for Hematologic Research, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
aAddress correspondence to this author at: Thomas Jefferson University, Medical Office Bldg, Room 406, 1100 Walnut St., Philadelphia, PA 19107. Fax 215-503-2803; e-mail paolo.fortina{at}mail.tju.edu.
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Abstract |
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Methods: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, ß-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with ß-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products.
Results: Analysis of amplified DNA required 4–6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods.
Conclusions: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Microelectronic array technology was recently introduced for SNP analysis (11)(12)(13)(14)(15). With the NanoChip® Molecular Biology Workstation, target oligomers and samples are electronically deposited on a proprietary cartridge composed of a 10 x 10 array of microelectrodes. A thin hydrogel permeation layer containing streptavidin coats the chip surface, allowing binding of the biotinylated PCR amplicon. Stabilizers and Cy5- and Cy3-labeled oligonucleotide reporters for each allele are hybridized, and the chip is then washed and imaged. Fluorescence signal ratios of the reporters allow discrimination between homozygotes and heterozygotes for a particular SNP. The technology provides either the amplicon-down format (biotinylated single-stranded PCR product from genomic DNA is chip bound) or the capture-down format (oligonucleotide proximal to the SNP is chip bound) (16)(17)(18)(19).
There are few published detailed protocols available for reference in designing custom assays for the SNPs frequently measured in a clinical laboratory. The SNPs/mutations assessed in this report include those involved in coagulation disorders, ß-thalassemia, and tumorigenesis. Three SNPs are located in the factor VII (FVII) gene as reported previously (20), including -402(G/A), -401(G/T), and -122(T/C), in addition to a polymorphic amino acid change (R353Q) and a decanucleotide insertion at -323 (insCCTATATCCT) (21). Mutations assessed in the ß-globin gene include IVS-I nt 1 (G/A), IVS-I nt 6 (T/C), and IVS-II nt 1 (G/A) (22)(23)(24), whereas those in the RET gene include C634G, C634R, C634Y, and M918T (25)(26)(27)(28)(29)(30). In this report, details of these assays are provided with advice to facilitate rapid development of other clinically important applications. Finally, protocols are provided with observations that are generalizable to other assays using this platform, thereby facilitating the rapid development of other clinically important applications.
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Materials and Methods |
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primers, stabilizers, and reporters
Gene sequences were obtained from GenBank (www.ncbi.nlm.nih.gov). Sequences for forward and reverse PCR primers, reporters, and stabilizers are detailed in Table 1
. All oligonucleotides were synthesized by IDT and purified by polyacrylamide gel electrophoresis and/or reversed-phase HPLC. Primers were designed to amplify the genomic region surrounding each SNP/mutation and were synthesized with one primer (forward or reverse) containing a 5'-biotinylated tag. Oligonucleotide reporters for the wild-type allele contained a 5'-Cy3 fluorophore, whereas the variant/mutant reporters contained a 5'-Cy5 fluorophore, except reporters for SNPs 5' of the FVII gene located at -401 and -402. For these two SNPs, two different stabilizer oligonucleotides were used for each SNP to be able to detect both in the same reaction. The two stabilizers for the -402 SNP differed at their 5' termini, whereas those for the -401 SNP differed at their 3' termini (Table 1
). Reporter oligonucleotides were labeled at the 5' end with Cy5 for the -402 SNP and were labeled at the 3' end with 6-carboxytetramethylrhodamine (6-TAMRA) for the -401 SNP instead of Cy3 to distinguish between the two alleles.
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pcr amplification
The following protocol was used for PCR amplifications encompassing the FVII and ß-globin genes. Genomic DNA samples were amplified by PCR in a 50-µL reaction containing 50 ng of genomic DNA, 2.5 mM MgCl2, 200 µM deoxynucleotide triphosphates, 300 nM each primer (one biotinylated), 2.0 U of AmpliTaq Gold DNA polymerase (PE Biosystems), and 1x PCR buffer II (PE Biosystems). Samples were denatured at 95 °C for 10 min and then cycled at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s for 35 cycles with a final extension at 72 °C for 5 min in a 9600 Thermocycler (PE Biosystems).
For the RET protooncogene, PCR conditions were as follows: 50 ng of DNA, 500 nM each primer, 1x PCR buffer II, 1.5 mM MgCl2, 200 µM deoxynucleotide triphosphates, and 0.05 U/µL AmpliTaq Gold DNA polymerase in a final volume of 50 µL. The RET protooncogene was amplified after denaturation at 95 °C for 10 min; cycling was as follows: 95 °C for 30 s, 55 °C for 20 s, and 72 °C for 40 s for 30 cycles, followed by a 5-min final extension. PCR products were purified and desalted on QIAquick spin columns (Qiagen) or with the Multiscreen Separation System (Millipore) with doubly distilled H2O used for the eluent. Desalting was performed for optimal performance of the electronic addressing of the samples to the chip (see below). QIAquick purification columns usually required one or more additional washes as recommended by Nanogen. Products were separated by gel electrophoresis and stained with ethidium bromide; PCR products were quantified by comparison of band intensities with MspI-digested pBR322 DNA. Amplified, biotinylated PCR product was incubated at a final concentration of 5–40 nmol/L with 250 nmol/L oligonucleotide stabilizer in 50 mmol/L histidine buffer (Sigma), heat-denatured for 5 min at 95 °C, cooled on ice, and transferred to a 96-well plate for the loading step. More recently, NaOH (0.1 mol/L) was used to denature PCR products in preparation for binding of single-stranded biotinylated products to streptavidin-containing gel pads.
microchips and instrumentation
Fabricated NanoChip cartridges were manufactured by Nanogen, Inc. Electronic addressing, processing, and scanning were performed on the NanoChip Molecular Biology Workstation, which consists of Loader and Reader components. Details of chip microfabrication, the instrumentation, and application of the permeation layer have been described previously (14)(15).
electronic addressing and hybridization
A sample-containing microtiter plate and a cartridge-containing microarray chip were placed together in the Loader drawer. Each assay was individually customized by assigning a specific position for each sample to be addressed on the chip, which contained a 99-pad map in a 10 x 10 configuration (1 pad is used as a reference electrode and cannot be used). Single-stranded biotinylated amplicons were electronically addressed (2.0 V for 120 s) to specific positively charged test sites on the NanoChip cartridge at room temperature. Sites also were addressed with histidine alone to serve as background controls. The cartridge was washed with 50 mmol/L histidine buffer after each address to remove unattached amplicons. Each sample was loaded in duplicate on two different pads, and a blank control (a PCR mixture with no DNA template) as well as a heterozygous control were included in each assay. Before starting the loading protocol, the loader automatically performed a 5-min conductivity test to check for activation of all 100 microelectrodes. In addition, the conductivity of the histidine buffer and each purified amplicon was checked by the conductivity meter supplied by Nanogen. Samples with a conductivity not exceeding 100 µS/cm were passed for loading.
Although we have not directly measured the failure rate for the sample loading process, the "Activation Test" from the manufacturer’s user guide shows the activation values for each addressing step. Low values can be caused by absence of DNA on the pads and can be used as an indication of failure in the loading procedure, which is sometimes caused by poor PCR product or quality of the buffers. High values may be an indication of too much salt on the chip caused by improper desalting. Our experience indicates that this process is highly efficient when good-quality PCR products are used.
Cy3- (wild-type) and Cy5- (mutant) labeled allele-specific oligonucleotide reporters were then mixed at 0.5 µmol/L each in high-salt buffer (50 mmol/L sodium phosphate, pH 7.4, and 500 mmol/L NaCl) and hybridized to the chip for 
3 min at room temperature as recommended by the manufacturer; the chip was then washed and scanned. Details of the washing and fluorescence detection steps are provided in the workstation manual. Increments of 2 °C were used to increase wash stringency until signals from wild-type and mutant probes were approximately the same in the heterozygous controls.
washing and fluorescence detection
After hybridization, the chip was washed in high-salt buffer (50 mmol/L sodium phosphate, pH 7.4, and 500 mmol/L NaCl), and an initial fluorescence scan was taken. Subsequently, to achieve precise discrimination between matched and mismatched alleles, increased stringency washes were done in low-salt buffer (50 mmol/L sodium phosphate, pH 7.0) under optimized temperature conditions (25–38 °C). Wash temperatures were gradually increased, depending on the reporter melting temperature (Tm). Chips were scanned for both fluorochromes after each wash at medium photomultiplier tube gain with an accumulation value of 256 as default settings for the laser. Background control values were subtracted from each pad’s signal for final quantification.
data analysis
Analyses were performed with dedicated automated software, provided from the company, that calculates the fluorescence values and genotype for each sample. In this analysis, values from sites addressed with either histidine alone or blank control (PCR mixture with no DNA template) were used for background subtraction, whereas the fluorescence values from the heterozygous control were used to normalize the Cy3/Cy5 signals to a value of 1. The normalized, background-subtracted mean values for each fluorochrome were then compared in each sample, and the dye ratios were calculated. As default settings recommended by the manufacturer, if the ratio of red (Cy5) to green (Cy3) was between 3:1 and 1:3, the sample was designated as heterozygous; ratios above 1:5 and 5:1 were designated as homozygous for the wild-type and mutant alleles, respectively, whereas ratios between 1:3 and 1:5 or between 3:1 and 5:1 indicated that the test should be repeated. The analysis program also considered a signal-to-noise ratio of 5:1 as a default value. Tests sites with a signal-to-noise ratio <5:1 were excluded from the analysis; however, this rule could be changed according to the specific signal values obtained in each assay. Red-to-green ratio default threshold values were not changed, as recommended by the manufacturer.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the Data Supplements available with the online version of this article at http://www.clinchem.org/vol48/issue12/). For each locus, we applied thermal stringency until the mismatched controls were removed to background values. In these plots, red (mutant) and green (wild-type) bars show typical data for heterozygotes as well as homozygotes for mutant or wild-type alleles. Quantification of the fluorescence emitted from each sample indicated a signal-to-noise ratio >5, thus allowing complete discrimination between the matched and mismatched reporter probes. Samples that produced a "no call" (8–12%) were retested after repurification of the PCR product. For some of the samples, retesting produced correct genotyping, whereas others remained a "no call" and were not pursued. Results are presented for analyses of SNPs in the FVII gene as an example; data for typing of ß-thalassemia and RET oncogene mutations are available as Data Supplements with the online version of this article.
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fvii
Activated FVII helps initiate clotting (31), and increased FVII is associated with coronary artery disease (32)(33)(34)(35)(36). A SNP in the FVII coding region at position 10976 at amino acid 353 and a decanucleotide insert (CCTATATCCT) at -323 in the promoter are associated with decreased FVII concentrations (37). However, recently we showed that the -323 insert is correlated with decreased FVII only when linked to a SNP at -122 (38).
The locations and sequences of SNPs associated with the FVII gene studied in this report are diagrammed in Fig. 1A
, with genotyping results presented for the SNPs at -401, -323, and -122 shown in Fig. 2
. With this approach, a known heterozygote is typed first on the workstation, and values for the green (wild-type) and red (mutant) probes are each normalized to a value of 1 (see the results for samples ET-246, 38A, and 36A; the last samples on right in Fig. 2
). Typing for the -122 SNP (right-hand panel) gave dye intensity ratios for wild-type (green) to mutant (red) targets of 8.34–13.62 in the seven wild-type homozygotes and ratios of 0.63–0.82 in the five heterozygotes. Unambiguous typing of all three genotypes is seen in the typing of the -323 polymorphism (middle panel) in 12 samples with red (deletion) to green(insertion) ratios of 5.0–20.45 for the seven homozygotes (deletion/deletion), 0.85–1.14 for the four heterozygotes, and 0.09 for the one homozygous mutant. Of the other 12 individuals typed relative to the known control heterozygote for the -401 SNP (left-hand panel), 10 were wild type/wild type (N/N), and 2 were heterozygotes. Typing was unambiguous, with corrected dye intensity ratios for hybridization of dye-tagged wild-type (green) to mutant (red) targets of 6–8.21 in wild-type homozygotes and ratios of 0.67–0.74 for heterozygotes. No homozygous mutants for the -401 SNP were available for typing.
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ß-globin gene
Decreased concentrations of ß-globin protein give rise to ß-thalassemia, which causes worldwide morbidity and mortality. More than 360 mutations are known (28)(29), with specific mutations being associated with particular ethnic groups (Fig. 1B
). For example, five mutations cause the majority of cases in the Mediterranean basin. These include mutations at IVS-I nt 1, IVS-I nt 6, IVS-I nt 110, IVS-II nt 1, and codon 39. We have typed mutations in IVS-I and -II associated with splice defects, which lead to decreased production of ß-globin RNA, on the Nanogen Workstation. Our results indicate that this platform clearly distinguishes heterozygotes for IVS-I nt 1 and nt 6 as well as IVS-II nt I from wild-type individuals (screen dumps in Data Supplements at www.clinchem.org/content/vol48/issue12/). To date, >600 samples have been genotyped for ß-thalassemia mutations with this platform, and those results were in complete concordance with previous, more traditional genotyping approaches (i.e., DNA sequence analysis and allele-specific PCR).
ret protooncogene
The RET protooncogene encodes a transmembrane tyrosine kinase receptor that may play a critical role in initiation of neuroblastomas, medullary thyroid carcinomas, and pheochromocytomas (25). In addition, mutations in RET have been identified in Hirschsprung disease (26)(28). Moreover, germline RET mutations are associated with the three variants of the inherited cancer syndrome, multiple endocrine neoplasia type 2 (MEN2A, MEN2B, and FMTC). The majority of MEN2A and FMTC cases involve mutations in the Cys-rich domains of exons 10 and 11, whereas 98% of patients with MEN2B have a transition involving codon 918 (ATG
ACG) in exon 16 of the RET protooncogene (M918T) (30) (Fig. 1C
). Three mutations at codon 634 (Cys to Gly, Arg, or Tyr) in exon 11 and a Met-to-Thr change at codon 918 in exon 16 were studied on this platform (screen dumps of data available on request). Typing for C634R in exon 11 and M918T in exon 16 confirmed previous genotyping of four wild-type individuals and six heterozygotes for the codon 634 change, and eight wild-type individuals and two heterozygotes for the codon 918 change.
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Discussion |
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In this study, we present details of some of the protocols developed to support our ongoing research activities, and we believe that they will minimize development time for adapting these techniques in other laboratories. In addition, we show that the microelectronic array technology is accurate in facilitating clinically important analyses.
In all samples analyzed with this allele-specific, gel pad-bound, single-stranded amplicon approach, we found 100% concordance with genotypes determined previously by restriction endonuclease digestion and/or DNA sequence analysis. We did find that 8–12% of samples produced "no calls", which were found to be the result of improperly prepared and/or insufficient amounts of PCR products. These samples were retested after repurification of the PCR product. Some were correctly genotyped on retesting, whereas others remained a "no call" and were not pursued. The flexibility of the system allowed the simultaneous use of a wide variety of probes of different content, length, and chemical composition on the same chip.
The protocols described are for detection of SNPs/mutations of common interest to the clinical research laboratory. Although these methods involve investment in initial development and optimization, the platform facilitates accurate genotyping and the potential for high-throughput analysis. Gel electrophoresis, restriction fragment length polymorphism analysis, passive probe hybridization, and DNA sequencing, although generally accepted as the current standard for mutation detection, are often laborious and time-consuming. The protocols we present should provide a vehicle to enable the potential of microelectronic array methods to be realized more quickly in a clinical laboratory setting, facilitating high-throughput, cost-effective molecular diagnostic testing of genetic variations as well as mutation detection.
Accuracy is of paramount importance in any genotyping platform, and our experience with this platform indicates that there are numerous advantages as well as areas of concern in using such a platform. Improvements could be made in the time required for analysis. The whole analytical process (excluding the 2 h required for PCR amplification) was performed in 4–6 h. This includes PCR product loading, probe hybridizations, washing, and hybrid detection. The longest time (3.5 h) was required for addressing biotinylated PCR products to the gel pads. If this process were streamlined, the entire time needed for typing could be greatly decreased because after the PCR products are addressed to the pads, the remaining steps are completed in minimum time. After addressing, Cy3- (wild-type) and Cy5- (mutant) labeled allele-specific oligonucleotide reporters are mixed in high-salt buffer and hybridized to the chip for 3 min at room temperature; the chip is then washed and scanned.
The advantages of this platform include the electronic addressing of single-stranded PCR products to specific array sites, which accelerates binding >1000-fold compared with traditional passive diffusion methods for deposition (16)(18). In addition, DNA is concentrated by charge attraction only to predefined sites on the array, greatly facilitating efficient deposition of amplicons. The ability to define which amplicons go where on the array affords the user flexibility in determining what loci are analyzed and facilitates rapid addition or deletion of loci to be typed. Use of electronic stringency affords the ability to rapidly define conditions that distinguish between perfect matches and single-base mismatches, thereby improving the accuracy of typing. Furthermore, the method provides flexibility during the hybridization and washing steps to enable delineation of perfect vs mismatched annealing. Hybridization times are generally much longer with passive diffusion compared with the electronically enhanced approach inherent in this platform. Multiple sites can be interrogated on the same array with the same patient material, thereby increasing throughput and providing a more cost-effective approach to typing.
An additional factor for consideration is cost per assay. After a one-time hardware purchase, typing costs for chips are estimated as approximately US $1.50 to $3.00 per sample plus technician time and reagents. Reagent costs are estimated to be minimal, and cost savings could be realized by maximizing the number of SNPs typed per chip or by minimizing the number of dye-tagged solution-phase reporters required. The former could be accomplished by increasing the number of addressable sites per chip, whereas the latter could be realized by use of a universal reporter strategy. This approach incorporates M13 or other scrambled sequences into the wild-type and mutant oligonucleotide reporters, which are then detected after allele-specific hybridization to chip-bound, single-stranded amplicons by complementary M13 dye-tagged wild-type or mutant detectors. In this way, dye-tagged oligonucleotides could be kept at a minimum of two, regardless of the number of loci to be typed.
In conclusion, the NMW 1000 NanoChip Molecular Biology Workstation appears useful for the development of analytical assays to identify SNPs/mutations in genes of medical importance. This platform offers advantages over others and can be readily implemented in a clinical laboratory setting with minimum effort to accomplish accurate, high-throughput SNP/mutation typing.
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Acknowledgments |
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Footnotes |
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References |
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-thalassemia repository (Ninth Edition; Part I). Hemoglobin 1998a;22:169-195.[Web of Science][Medline]
[Order article via Infotrieve]
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