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Technical Briefs |
1 Department of Clinical Biochemistry, Herlev University Hospital, Copenhagen, Denmark;2 Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, Copenhagen, Denmark
aaddress correspondence to this author at: Department of Clinical Biochemistry, 54M1, Herlev University Hospital, Herlev Ringvej 75, DK-2730 Copenhagen, Denmark; fax 45-4488-3311, e-mail brno{at}herlevhosp.kbhamt.dk
To increase the speed and reduce the cost of single-nucleotide polymorphism (SNP) genotyping, we evaluated a new potential high-throughput genotyping method, the NanogenTM NMW 1000 NanochipTM Molecular Biology Workstation (1). We tested the Nanogen microelectronic chip for SNP genotyping with respect to throughput, accuracy, and cost-effectiveness. To increase throughput we first developed two different multiplex methods by which three different SNPs are determined sequentially at each test site. We then validated accuracy by genotyping 3637 individuals for three SNPs, using the microelectronic chip as well as restriction fragment length polymorphism (RFLP) analysis. Finally, we compared the cost-effectiveness of SNP genotyping by the Nanogen microelectronic chip and RFLP and evaluated the reuse of microelectronic chips to decrease the cost per SNP genotyped.
As a first step, we developed two different protocols for multiplex SNP analyses: (a) PCR amplification of 557 bp of the ß2-adrenergic receptor gene, including three known SNPs detected sequentially at each test site, and (b) multiplex PCR to amplify three regions of the hepatic lipase gene, each with one known SNP, addressed simultaneously and detected sequentially at each test site.
To test accuracy we next genotyped three ß2-adrenergic receptor SNPs in 3637 individuals from the Danish general population, The Copenhagen City Heart Study (2)(3)(4), using Nanogen microelectronic chips as well as RFLP analysis. Any samples showing discordance between the two methods were sequenced.
We then compared total running cost (supplies and wages) when SNPs were genotyped with Nanogen microelectronic chips and by RFLP analysis. In addition, in 376 individuals we compared genotype outcome for three hepatic lipase SNPs between four new microelectronic chips and four microelectronic chips already used to genotype the three SNPs in the ß2-adrenergic receptor gene.
The Danish ethics committee for Copenhagen and Frederiksberg approved this study (No. 100.2039/91), and all participants gave informed consent.
Because of the very low fluorescence signals from the Gln27Glu and Thr164Ile reporters, we speculated that hairpins probably blocked hybridization of reporters with the single-stranded PCR fragment (Fig. 1
). To improve the fluorescence signals of reporters, we added one extra stabilizer for Gln27Glu (Tables 1 and 2 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue2/) and two extra stabilizers for Thr164Ile (Tables 1 and 2 in the online Data Supplement), which neutralized potential hairpins and gave higher fluorescence signals for all reporters. Among 3637 individuals genotyped, the allele frequencies for Arg16, Glu27, and Ile164 were 37.4%, 43.8%, and 1.6%, respectively.
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SNPs in the hepatic lipase gene that produced the substitutions Asn193Ser, Ser267Phe, and Leu334Phe were genotyped by simultaneous addressing of three small PCR fragments (Table 1 in the online Data Supplement) with one SNP each followed by sequential detection at each test site. Among 5625 individuals genotyped, allele frequencies for Ser193, Phe267, and Phe334 were 36.6%, 0.4%, and 1.7%, respectively.
Three SNPs in the ß2-adrenergic receptor gene that produced the substitutions Arg16Gly, Gln27Glu, and Thr164Ile were determined in a total of 3637 individuals (10 911 determinations) by both the microelectronic chip and RFLP analysis. A total of nine determinations were true misclassifications for Arg16Gly or Gln27Glu (Table 2 in the online Data Supplement). Sequencing suggested three misclassifications (0.03%) by RFLP analysis and six by the microelectronic chip (0.05%; P = 0.32). After the misclassified samples were reanalyzed, all genotypes were in accordance with the sequencing result. It is therefore likely that the misclassification of three SNPs by RFLP analysis and six by microelectronic chips was attributable to sample mix-up. RFLP analysis had more "no calls" than the microelectronic chip (349 vs 234 of a total 3637; P <0.001).
The total running costs per SNP genotyped in the ß2-adrenergic receptor gene by the new microelectronic chips and RFLP analysis were 
1.10 and 0.88 (Table 1
). Time consumed by technicians (i.e., hands on) for genotyping of all three SNPs were the same for the two methods; however, we observed a difference in the cost of supplies, mainly attributable to the cost of microelectronic chips.
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The results obtained for hepatic lipase SNP determinations on chips previously used to genotype ß2-adrenergic receptor SNPs were comparable to the results obtained for SNP determinations on new microelectronic chips (n = 376 individuals and 1128 genotypes). During the initial analysis of samples, 87% of the genotypes were correct with both new and previously used chips; among reruns, correct genotypes were obtained for 97% and 79% of the samples analyzed on new and reused chips, respectively. Total running cost per SNP genotyped for reused microelectronic chips was 0.40 (Table 1
).
In summary, we examined a new high-throughput genotyping system based on a microelectronic chip assay. We demonstrated (a) that the two different multiplex microelectronic chip assays enable high-throughput SNP analysis, (b) that accuracy for the microelectronic chip and standard RFLP analysis was comparable, and (c) that reuse of microelectronic chips substantially lowered the cost per SNP genotyped without loss of accuracy.
The microelectronic chip has been shown to be flexible for high-throughput genotyping: it enabled us to genotype three SNPs per test site (e.g., per individual) in two very different and distinct ways. We first demonstrated detection of individual SNPs located in one large PCR fragment of the ß2-adrenergic receptor gene; we then demonstrated SNP detection when SNPs were located in different exons of the hepatic lipase gene by use of multiplex PCR.
Our results imply that the microelectronic chip has greater flexibility (using the same DNA chip format for different SNP genotyping assays) than other microarray/chip methods, which are limited to genotyping of a standard predesigned set of SNPs (5). Other studies have also shown the multiplexing ability of the microelectronic chip by analyzing eight different SNPs on one test site simultaneously (6). Furthermore, compared with other microarray systems, the microelectronic chip allows simultaneous use of a wide variety of probes of different lengths and chemical compositions on the same chip. The microelectronic assay also allows different stringency conditions to be applied at each site of the chip, allowing the use of optimum conditions for each of the consecutive hybridization reactions on the chip (1). In contrast, on standard microarray chips, common reaction and stringency conditions have to be applied to all addresses.
Previous reports validating the accuracy of microelectronic chips by comparing them with RFLP analysis and sequencing included only 22–83 individuals, and the authors found total concordance between the microelectronic chip and other genotyping methods (7)(8)(9). In our hands, the number of genotyping errors also were comparable for the two methods (0.05% vs 0.03%; P = 0.32), but RFLP analysis produced more no calls than the microelectronic chip. The few misclassified genotypes on the microelectronic chip could be attributable to sample mix-up because reanalysis of the six misclassified SNPs gave results identical to the sequencing results.
The main expense of genotyping by the microelectronic chip is the chip itself (72% of cost of supplies). Maximizing the number of SNPs typed per chip could minimize this cost. We therefore tested the possibility of SNP detection on previously used chips and found that previously used microelectronic chips could be reused for detection of new SNPs in other genes without loss of accuracy; however, there was substantial falloff in terms of performance with new vs used chips. This lowers the cost per SNP to almost one-third of the cost of using a new microelectronic chip and more lowers the cost compared with RFLP analysis by more than one-half. This is approximately the same cost reported by others for detecting eight SNPs on one test site simultaneously (1.62 per SNP) (6). It should be noted, however, that purchase of the Nanogen NMW 1000 Nanochip Molecular Biology Workstation is not included in these calculations.
The microelectronic chip examined in this study was limited by the number of test sites per chip. Increasing the number of addressable sites per chip to 400 (7) would improve throughput. Until this is achieved, the microelectronic chip is potentially useful primarily when many SNPs can be detected on the same test site. This would minimize the cost per SNP and contribute to high throughput. Advantages compared with other microarray methods include the possibility of reusing chips and the flexibility, which allows each chip to be customized according to the relevant assay design.
Although other DNA microarray systems can simultaneously detect many SNPs in one individual, genotyping is limited because of the need to use predefined sets of SNPs (5), the low accuracy of heterozygous samples (10), and the high cost. Fluorescence resonance energy transfer also has high-throughput capabilities but requires large amounts of DNA (
50 ng) per SNP determination and lacks multiplexing ability compared with the microelectronic chip (11)(12). The TaqMan method, which also is capable of high throughput, has no intermediate processing, making the method highly automated compared with the microelectronic chip (13). Although mass spectrometry is a high-throughput genotyping system, it requires, like the microelectronic chip, high-purity samples for genotyping, thus increasing technician time and sample-processing costs (14)(15).
In conclusion, we demonstrate that SNP detection by microelectronic chips is comparable to RFLP analysis in terms of throughput, accuracy, and cost-effectiveness, but the limitations of microchip analysis, such as the high purchase price and lower amenability to automation, must be kept in mind.
Acknowledgments
We thank Birgit Hertz, Hanne Damm, Vibeke Wohlgehagen, Nina Dahl Kjersgaard, and Anja Jochumsen for expert technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  D. D. Orsted, S. E. Bojesen, A. Tybjaerg-Hansen, and B. G. Nordestgaard Tumor suppressor p53 Arg72Pro polymorphism and longevity, cancer survival, and risk of cancer in the general population J. Exp. Med., June 11, 2007; 204(6): 1295 - 1301. [Abstract] [Full Text] [PDF]
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 M. Sanguinetti, L. Novarese, B. Posteraro, S. Ranno, E. De Carolis, G. Pecorini, B. Lucignano, F. Ardito, and G. Fadda Use of Microelectronic Array Technology for Rapid Identification of Clinically Relevant Mycobacteria J. Clin. Microbiol., December 1, 2005; 43(12): 6189 - 6193. [Abstract] [Full Text] [PDF]
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