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However, There Was Substantial Falloff in Terms Of 446 Technical Briefs accuracy; however, there was substantial falloff in terms disease, myocardial infarction, and ischemic cerebrovascular disease. Six case-control studies from the Copenhagen City Heart Study. Ann Intern Med of performance with new vs used chips. This lowers the 2001;134:941–54. cost per SNP to almost one-third of the cost of using a new 5. Twyman RM, Primrose SB. Techniques patents for SNP genotyping. Pharma- microelectronic chip and more lowers the cost compared cogenomics 2003;4:67–79. 6. Thistlethwaite WA. Rapid genotyping of common MeCP2 mutations with an with RFLP analysis by more than one-half. This is approx- electronic DNA microchip using serial differential hybridization. J Mol Diagn imately the same cost reported by others for detecting 2003;5:121–6. eight SNPs on one test site simultaneously (€1.62 per SNP) 7. Gilles PN, Wu DJ, Foster CB, Dillon PJ, Chanock SJ. Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor (6). It should be noted, however, that purchase of the microchips. Nat Biotechnol 1999;17:365–70. Nanogen NMW 1000 Nanochip Molecular Biology Work- 8. Nagan N, O’Kane DJ. Validation of a single nucleotide polymorphism station is not included in these calculations. genotyping assay for the human serum paraoxonase gene using electroni- cally active customized microarrays. Clin Biochem 2001;34:589–92. The microelectronic chip examined in this study was 9. Sohni YR, Dukek B, Taylor W, Ricart E, Sandborn WJ, O’Kane DJ. Active limited by the number of test sites per chip. Increasing the electronic arrays for genotyping of NAT2 polymorphisms. Clin Chem 2001; number of addressable sites per chip to 400 (7) would 47:1922–4. 10. Shi MM. Enabling large-scale pharmacogenetic studies by high-throughput improve throughput. Until this is achieved, the microelec- mutation detection and genotyping technologies. Clin Chem 2001;47:164– tronic chip is potentially useful primarily when many 72. 11. Lyamichev V, Mast AL, Hall JG, Prudent JR, Kaiser MW, Takova T, et al. SNPs can be detected on the same test site. This would Polymorphism identification and quantitative detection of genomic DNA by minimize the cost per SNP and contribute to high invasive cleavage of oligonucleotide probes. Nat Biotechnol 1999;17: throughput. Advantages compared with other microarray 292–6. 12. Tsuchihashi Z, Dracopoli NC. Progress in high throughput SNP genotyping methods include the possibility of reusing chips and the methods. Pharmacogenomics J 2002;2:103–10. flexibility, which allows each chip to be customized 13. Livak KJ, Marmaro J, Todd JA. Towards fully automated genome-wide according to the relevant assay design. polymorphism screening. Nat Genet 1995;9:341–2. 14. Ross P, Hall L, Smirnov I, Haff L. High level multiplex genotyping by Although other DNA microarray systems can simulta- MALDI-TOF mass spectrometry. Nat Biotechnol 1998;16:1347–51. neously detect many SNPs in one individual, genotyping 15. Griffin TJ, Smith LM. Single-nucleotide polymorphism analysis by MALDI-TOF is limited because of the need to use predefined sets of mass spectrometry. Trends Biotechnol 2000;18:77–84. SNPs (5), the low accuracy of heterozygous samples (10), and the high cost. Fluorescence resonance energy transfer DOI: 10.1373/clinchem.2003.026047 also has high-throughput capabilities but requires large amounts of DNA (ϳ50 ng) per SNP determination and lacks multiplexing ability compared with the microelec- tronic chip (11, 12). The TaqMan method, which also is Rapid Quantification of DNase I Activity in One-Micro- capable of high throughput, has no intermediate process- liter Serum Samples, Haruo Takeshita,1† Tamiko Naka- ing, making the method highly automated compared with jima,1† Kouichi Mogi,1 Yasushi Kaneko,1 Toshihiro Yasuda,2 the microelectronic chip (13). Although mass spectrome- Reiko Iida,3 and Koichiro Kishi1* (1 Department of Legal try is a high-throughput genotyping system, it requires, Medicine and Molecular Genetics, Gunma University like the microelectronic chip, high-purity samples for Graduate School of Medicine, Maebashi, Gunma 371- genotyping, thus increasing technician time and sample- 8511, Japan; Departments of 2 Biology and 3 Forensic processing costs (14, 15). Medicine, Fukui Medical University, Matsuoka, Fukui In conclusion, we demonstrate that SNP detection by 910-1193, Japan; † these authors contributed equally in microelectronic chips is comparable to RFLP analysis in this study; * author for correspondence: fax 81-27-220- terms of throughput, accuracy, and cost-effectiveness, but 8035, e-mail [email protected]) the limitations of microchip analysis, such as the high purchase price and lower amenability to automation, Deoxyribonuclease I (DNase I; EC 3.1.21.1) is genetically must be kept in mind. polymorphic (1), and has been postulated to be a candi- date molecule for the endonucleolytic activity involved in apoptosis or programmed cell death (2) and involved in We thank Birgit Hertz, Hanne Damm, Vibeke Wohlgeha- the initiation of systemic lupus erythematosus (3).We gen, Nina Dahl Kjersgaard, and Anja Jochumsen for have reported the presence of high DNase I activity and expert technical assistance. its gene expression in the anterior lobe of the pituitary gland of both sexes and the abrupt increase in its gene References expression at the onset of puberty (4). These findings 1. Sosnowski RG, Tu E, Butler WF, O’Connell JP, Heller MJ. Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field suggest that DNase I has other, unknown biological roles control. Proc Natl Acad Sci U S A 1997;94:1119–23. as well as a digestive role. The single radial enzyme 2. Schnohr P, Jensen G, Lange P, Scharling H, Appleyard M. The Copenhagen diffusion (SRED) method (5, 6) has been used to quantify City Heart Study. Østerbroundersøgelsen. Tables with data from the third examination 1991–1994. Eur Heart J 2001;3:1–83. DNase I activity and allows the measurement of very low 3. Sethi AA, Nordestgaard BG, Agerholm-Larsen B, Frandsen E, Jensen G, DNase I activity in serum samples, although it requires a Tybjaerg-Hansen A. Angiotensinogen polymorphisms and elevated blood long incubation period (10–20 h). Recently we proposed pressure in the general population: the Copenhagen City Heart Study. Hypertension 2001;37:875–81. that determination of serum DNase I activity is useful in 4. Sethi AA, Tybjaerg-Hansen A, Gronholdt ML, Steffensen R, Schnohr P, the diagnosis of acute myocardial infarction (AMI) in the Nordestgaard BG. Angiotensinogen mutations and risk for ischemic heart early phase after onset (7). However, the present SRED Clinical Chemistry 50, No. 2, 2004 447 assay takes too long to be useful in the emergency room, which the sample was applied and digested the DNA and the development of a more rapid assay for DNase I is substrate. Incubation continued until test samples showed desirable for therapeutic decision-making. In this report, well-defined dark circles with diffusion diameters (d)of we describe a novel assay method that can be used to 2.0–15.0 mm. Differences in length were measured in quantify DNase I activity in 1-␮L serum samples within 0.1-mm increments. A calibration curve was constructed 30 min. by plotting log10 DNase I activity against d. Human DNase I was purified from urine as described The SRED/CAM method is based on the fact that SG previously (8). Antibodies specific to human DNase I and shows fluorescence only with unhydrolyzed DNA and II were produced in rabbits (8–10). Cellulose acetate not with DNA digested by DNase I (5, 6). A dark circular membrane (CAM) was purchased from Sartorius, SYBR zone is formed on the fluorescent background as DNase I Green I (SG) was from Cambrex, and salmon testicular diffuses from the sample spot position into the CAM sheet DNA (type III) was from Sigma. containing DNA, SG, divalent cations, and buffer and An assay reagent set for DNase I activity was prepared subsequently hydrolyzes DNA (Fig. 1, inset). We found in two steps: production of a gel plate for keeping the that the d was linearly proportional to the logarithm of Ϫ CAM wet, and preparation of CAM containing reaction DNase I activity within the range of 0.01 to 50 ϫ 10 5 U, buffer. In step 1, 20 mL of 5 g/L molten agarose GP-36 corresponding to a range of 0.05–250 pg calculated from (Nacalai Tesque) in distilled water at 55 °C was poured the specific activity of purified human DNase I after a into a horizontal plastic tray (7.5 ϫ 14.0 cm; 1.0 cm deep) 15-min incubation. Longer incubation times of 30 and 45 with a lid (EIKEN). After solidification at room tempera- min increased sensitivity 1.7- and 2.3-fold, respectively Ϫ ture, the lid was placed on the gel dish, and the dish was (Fig. 1). The lower limit of detection was 0.5 ϫ 10 8 U for stored at 4 °C. In step 2, we prepared two working both the 30- and 45-min incubations. The upper limit of Ϫ solutions. For the first solution, we dissolved 10 g/L of linearity was 2 ϫ 10 4 U for all three incubation times (15, salmon testicular DNA in distilled water, stirred it for 2–3 30, and 45 min). h, and stored the solution at 4 °C. For the second solution, The within- and between-run imprecision studies were we diluted the SG 250-fold with dimethyl sulfoxide. To performed with the buffer described above and purified prepare the reaction buffer, we mixed the SG (1.0 ␮L) and human DNase I at two selected concentrations (at the low DNA (0.4 mL) solutions and buffer [9.6 mL, containing 0.1 and high ends of the assay).
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