Clinical Chemistry 46, No. 11, 2000 of sample containing multiple components (pBR322/MspI plasmid digest; Sigma) in even-numbered wells (chiploading protocol D). No interwell contamination was observed during chip analysis. Thus, the improved consistency of signal quantification observed was not attributable to interwell contamination but rather to enhanced intrawell sample mixing. Chip-loading protocol D was used for all subsequent experiments. To evaluate the ability of the Bioanalyzer to measure absolute concentrations, concentrated eNOS PCR product (⬃40 ng/␮L) was diluted in Tris-EDTA buffer to yield solutions containing 2.6, 5.1, 13.0, and 28.4 ng/␮L. Each sample was loaded twice on each chip, with one sample being loaded in wells 1– 6 and a duplicate sample being loaded into wells 7–12. Concentration values generated by the Bioanalyzer differed from concentration values obtained spectrophotometrically by 6 –16%. Well-to-well and chip-to-chip results differed by similar amounts. DNA sizing results were found to be independent of the cDNA concentration. To evaluate the ability of the Bioanalyzer to size samples containing multiple DNA fragments, commercially available plasmid digests were analyzed. To reduce the concentration of DNA fragments ⬍100 bp in size, plasmid digests were spin-purified using the QIAquick PCR purification method, and samples were eluted in 1⫻ TrisEDTA buffer and diluted to yield appropriate concentrations for analysis. pUC18/MspI digest (Sigma) samples were loaded into wells 1, 4, 7, and 10; ␾X174 RF DNA/ HaeIII fragments (Gibco) samples were loaded into wells 2, 5, 8, and 11; ␭ DNA/EcoRI marker (Promega) samples were loaded into wells 3, 6, 9, and 12. This experiment was performed twice on each of 2 successive days. Table 1, D and E, shows sizing and signal quantification results, respectively, for ␾X174 RF DNA/HaeIII. The CV for DNA sizing of fragments was ⱕ2.1%, whereas the CV for DNA signal quantification was ⱕ6.7%. Similar CV values were observed for pUC18/MspI digest with respect to sizing and quantification. CVs for DNA sizing and quantification were ⬃8% and 7%, respectively, for ␭ DNA/EcoRI markers. Similar results were observed for unpurified plasmid digests. In summary, we recommend a modification to the manufacturer’s protocol for chip loading: namely, gentle pipetting of samples with the marker mixture after loading into the sample wells, followed by vortex-mixing for 1 min at the highest setting, which does not cause liquid loss from the sample wells. Lower concentration DNA fragments may not be detected if poor chip preparation leads to weak sample staining. This is particularly crucial with respect to the manufacturer-supplied molecular size ladder. The Bioanalyzer cannot calculate size and concentration values for the experimental samples if it fails to detect all bands in the ladder. In addition, improper staining of the upper molecular size marker may lead to poor quantification of experimental DNA fragments. We found the Agilent 2100 Bioanalyzer to be an easyto-use, time-efficient substitute to conventional CE. It was effective at sizing and quantifying multiple DNA frag- 1853 ments in a variety of experimental samples, including plasmid digests and PCR samples. This work was supported by grants from the NCI (Grant CA78848-02 to P.W. and L.J.K.) and NIH (Grant P60HL38632 to P.F.). P.W. and L.J.K. previously were recipients of grant support from Caliper Technology Corporation and were stock holders of and consultants to this company. References 1. Beckman Instruments, Inc. Introduction to capillary electrophoresis. Technical Bulletin 360643. Fullerton, CA: Beckman Instruments, 1994. 2. Mitchelson KR, Cheng J, Kricka LJ. The use of capillary electrophoresis for point-mutation screening. Trends Biotechnol 1997;15:448 –58. 3. Guttman A, Ulfelder KJ. Separation of DNA by capillary electrophoresis. Adv Chromatogr 1998;38:301– 40. 4. Dolnik V. Recent developments in capillary zone electrophoresis of proteins. Electrophoresis 1999;20:3106 –15. 5. Kasicka V. Capillary electrophoresis of peptides. Electrophoresis 1999;20: 3084 –105. 6. Smith JT. Recent advances in amino acid analysis using capillary electrophoresis. Electrophoresis 1999;20:3078 – 83. 7. Cheng J, Waters LC, Fortina P, Hvichia G, Jacobson SC, Ramsey JM, et al. Degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human DNA on microchip-based devices. Anal Biochem 1998;257:101– 6. 8. Jacobson SC, Ramsey JM. Microchip electrophoresis with sample stacking. Electrophoresis 1995;16:481– 6. 9. Waters LC, Jacobson SC, Kroutchinina N, Kondurina J, Foote RS, Ramsey JM. Microchip device for cell lysis, multiplex PCR amplification, and electrophoretic sizing. Anal Chem 1998;70:158 – 62. 10. Waters LC, Jacobson SC, Kroutchinina N, Kondurina J, Foote RS, Ramsey JM. Multiple sample PCR amplification and electrophoretic analysis on a microchip. Anal Chem 1998;70:5172– 6. 11. Khandurina J, Jacobson SC, Waters LC, Foote RS, Ramsey JM. Microfabricated porous membrane structure for sample concentration and electrophoretic analysis. Anal Chem 1999;71:1815–9. 12. Guttman A. Effect of operating variables on the separation of DNA molecules by capillary electrophoresis. Appl Theor Electrophor 1992;3:91– 6. 13. Marsden PA, Heng HHQ, Scherer SW, Stewart RJ, Hall AV, Shi X-M, et al. Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. J Biol Chem 1993;268:17478 – 88. Evaluation of a Nucleic Acid-based Cross-Linking Assay to Screen for Hereditary Hemochromatosis in Healthy Blood Donors, Christiane Wylenzek,1 Martina Engelmann,1 Dirk Holten,1 Reuel Van Atta,2 Michael Wood,2 and Birgit Gathof 1* (1 Division of Transfusion Medicine, University Hospital of Cologne, Joseph-Stelzmann Strasse 9, 50924 Cologne, Germany; 2 NAXCOR, 4600 Bohannon Dr., Suite 220, Menlo Park, CA 94025; * author for correspondence: fax 49-0221-478-6179, e-mail Birgit.Gathof@ medizin.uni-koeln.de) Hereditary hemochromatosis (HH) is a common autosomal recessive disorder (frequency, 1 in 300 –500 in the Northern European population) characterized by overabsorption of iron with consequent multiorgan failure secondary to iron overload (1, 2 ). Because early diagnosis and therapy can entirely prevent clinical complications, HH presents a model system for presymptomatic detection at the molecular level. HFE, the disease-causing gene of HH, encodes a 343-amino acid protein with high 1854 Technical Briefs structural similarity to MHC class I molecules (3, 4 ). The primary disease-causing mutation is a single G-to-A transition at position 845, encoding a protein with a Cys282Tyr amino acid substitution (3 ). Current HH genotyping techniques include restriction fragment length polymorphism (RFLP) analysis (5 ) and heteroduplex analysis (6 ), both PCR based. We evaluated a nucleic acid-based test with cross-linkable DNA probes to screen for the Cys282Tyr mutation in a total of 101 presumably healthy blood donors. The assay uses oligonucleotide probes modified with photo-activatable crosslinker molecules (7 ) and has been used to detect the factor V Leiden mutation (8 ). Two sets of allele-specific crosslinkable DNA probes were prepared that detect either the wild-type or mutant Cys282Tyr gene sequences. Samples prepared from donor blood were assayed with each probe set, and the genotype of each individual was determined by comparison of the fluorescent signals obtained. All blood samples were also assayed by a PCR-RFLP test. Two capture probes that hybridized preferentially to the wild-type or mutant HFE gene sequence, respectively, were synthesized for the cross-linking assay: (5⬘-3⬘) AXATACGTGCCAGGTG and AXATACGTACCAGGTGG (the underlined bases represent the position of the mutation site in the target). The coumarin-based cross-linking nucleotide is denoted as X; both probes were biotinylated at the 3⬘ end (8 ). In addition, 24 reporter probes were synthesized, each containing two fluorescein residues at the 5⬘ terminus and a cross-linker molecule in place of a nucleotide one position from the 3⬘ terminus, the 5⬘ terminus, or both. The reporter probes were designed from HFE gene sequence flanking the mutation, corresponding to the following nucleotide positions (9 ): 6443– 6462, 6472– 6493, 6495– 6518, 6574 – 6593, 6597– 6618, 6632– 6653, 6661– 6682, 6686 – 6707, 6716 – 6739, 6780 – 6799, 6801– 6822, 6838 – 6861, 6877– 6900, 6905– 6928, 6951– 6973, 6984 –7006, 7060 –7083, 7089 –7108, 7128 –7151, 7163–7186, 7208 –7231, 7235–7256, 7301–7322, and 7381–7402. Blood specimens were obtained from 101 blood donors with informed consent under an institutional review board-approved protocol (University of Cologne). Leukocytes were isolated from blood samples as described (8 ), resuspended in leukocyte lysis reagent (0.28 mol/L NaOH), and either boiled at 100 °C for 30 min immediately before the assay or stored at ⫺20 °C for up to 14 days before boiling. Processed samples were placed into two wells each of a 96-well polypropylene microtiter plate. Each assay plate also contained four negative controls (leukocyte lysis reagent that had not been boiled) and two positive controls (50 amol/well of a PCR amplicon covering the assay locus amplified from a Cys282Tyr and wild-type heterozygote in leukocyte lysis reagent that had not been boiled). Two different probe solutions were prepared, each containing the same set of 24 reporter probes and 1 of the 2 allele-specific capture probes. Aliquots of each probe solution were added to one of each pair of sample wells, as well as two negative and one positive control wells. Neutralization of the solutions, photo cross-linking, and addition of the streptavidin-coated magnetic beads have been described (8 ). The beads were then washed twice with wash reagent (0.15 mol/L NaCl, 0.015 mol/L sodium citrate, 1 mL/L Tween-20). The beads were incubated in the presence of anti-fluorescein antibody-alkaline phosphatase conjugate (Dako), washed four times, and resuspended in AttophosTM (Promega) as described (8 ). The fluorescence signal was determined by reading the plate in a microplate fluorometer (Packard Instrument). Genomic DNA was extracted from whole blood by the QIAquick Blood reagent set (QIAGEN). A sequence flanking the variant codon 282 was amplified by PCR (DyNAzyme PCR reagent set; Biometra), and the amplicons were digested with RsaI (Roche), size-fractionated by agarose gel electrophoresis, and genotyped as described (5 ). Determination of the genotype of an individual with the cross-linking assay was based on the relative signals obtained with the two allele-specific capture probe preparations. The net sample signal (NSS) was derived for each sample and each probe set by subtracting the mean of the negative control values from the sample signal. The NSS ratio was defined for each sample as the NSS for the mutation divided by the NSS for the wild type. The NSS ratio intervals that define a particular genotype were set before the donor samples were tested by assaying PCR amplicons derived from individuals who were wild type, heterozygous, or mutant homozygous for the Cys282Tyr allele (20 determinations for each genotype). These experiments yielded the following mean NSS ratios: wild type, 0.05 (range, 0.01– 0.45); heterozygous, 1.22 (range, 0.95– 2.15); and mutant homozygous, 6.13 (range, 2.95–15.00). On the basis of these results, the following NSS ratio intervals were used to assign a sample genotype: wild type, NSS ratio ⫽ 0 – 0.75; heterozygous, NSS ratio ⫽ 0.76 –2.5; and homozygous mutant, NSS ratio ⬎2.5. The sample data fell into two groups (Fig. 1). The first group of 93 samples had NSS ratios of 0.12– 0.66 (mean ⫽ 0.36; SD ⫽ 0.11) and was assigned a wild-type genotype. The second group (eight samples) had NSS ratios of Fig. 1. Frequency distribution of NSS ratios obtained by testing 101 blood donor samples with the cross-linking assay. The dotted line represents the cutoff between Cys282Tyr heterozygous and wild-type samples. Clinical Chemistry 46, No. 11, 2000 1855 1.02–1.55 (mean ⫽ 1.30; SD ⫽ 0.18), compatible with heterozygosity. No individuals homozygous for the Cys282Tyr mutation were identified. To validate the method for detection of the homozygous mutant genotype, the assay was performed on a blood sample from a known homozygote. The NSS ratios for this individual, in two evaluations, were 9.1 and 7.4, within the predicted range. The results of PCR-RFLP testing were in complete agreement with those obtained with the cross-linking assay for all 101 samples. The cross-linking assay has several advantages. It allows detection of the Cys282Tyr mutation without the laborious steps of DNA purification, PCR, and RFLP analysis, and it eliminates problems of sample inhibition of polymerases and sample contamination by amplicons. An additional advantage is the large-scale simultaneous processing of DNA samples, using the microtiter plate format. With automated detection, the cross-linking assay can be finished within 4 h. Further work is needed to fully define the set of NSS ratio ranges that determine the three genotypes. Data from the blood sample assays showed wider variation among samples of the same genotype than was seen with the PCR samples. Presumably, this indicates that signal intensity is influenced by factors such as the efficiency of the overall sample preparation procedure and variation in blood volume and leukocyte concentration. Further sample data will allow us to set finer intervals for genotype assignment and to set “gray zone” values for repeat testing. Large-scale, presymptomatic screening of blood donors for the Cys282Tyr mutation could identify individuals at risk for HH, who are then candidates for prophylactic phlebotomy, which increases the life expectancy to that of the general population. If such a screening regimen was to be implemented, the tests needed to perform genotype analysis will have to be accurate, inexpensive, and automatable. The cross-linking assay used here is an efficient, simple, and rapid method of genotyping HFE mutations that, with automation, would be suitable for routine genetic analysis in a large-scale manner. hybridization assay for direct detection of factor V Leiden mutation. Clin Chem 1997;43:1703– 8. 9. Albig W, Drabent B, Burmester N, Bode C, Doenecke D. GenBank Accession No. Z92910. National Center for Biotechnology Information. http://www. ncbi.nlm.nih.gov (accessed January 1999). References The human kallikrein gene family is important to the discipline of clinical chemistry because it contains genes that encode for valuable cancer biomarkers, including the best tumor marker available today, prostate-specific antigen (PSA). Despite reports of numerous kallikrein-like genes in the mouse (1 ), until 2–3 years ago, only three human kallikrein genes were recognized: pancreatic/ renal kallikrein (KLK1), human glandular kallikrein 2 (KLK2), and prostate-specific antigen (KLK3) (1, 2 ). The proteins encoded by the three kallikrein genes are now known as hK1, hK2, and hK3 (PSA). These three genes encode for serine proteases with either trypsin-like (hK1, hK2) or chymotrypsin-like (hK3) activity. Traditionally, kallikreins have been defined as enzymes that can act on high-molecular weight substrates and release bioactive peptides, known as kinins (3 ). Among the known kal- 1. Edwards CQ, Griffen LM, Goldgar D, Drummond C, Skolnick MH, Kushner JP. Prevalence of hemochromatosis among 11065 presumably healthy blood donors. N Engl J Med 1988;318:1355– 62. 2. McLaren C, Gordeuk V, Looker A, Hasselblad V, Edwards CQ, Griffin LM, et al. Prevalence of heterozygotes for hemochromatosis in the white population of the United States. Blood 1995;86:2021–7. 3. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary hemochromatosis. Nat Genet 1996;13:399 – 408. 4. Camaschella C, Piperno A. Hereditary hemochromatosis: recent advances in molecular genetics and clinical management. Haematologica 1997;82:77– 84. 5. Jouanolle AM, Fergelot P, Gandon G, Yaouanq J, Le Gall JY, David V. A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations. Hum Genet 1997;100:544 –7. 6. Jackson HA, Bowen DJ, Worwood M. Rapid genetic screening for haemochromatosis using heteroduplex technology. Br J Hematol 1997;98:856 –9. 7. Wood M, Albagli D, Cheng P, Huan B, Van Atta R. Nucleic acid crosslinking probes for DNA/RNA diagnostics [Abstract]. Clin Chem 1996;42:S196. 8. Zehnder J, Van Atta R, Jones C, Sussmann H, Wood M. Cross-linking New Nomenclature for the Human Tissue Kallikrein Gene Family, Eleftherios P. Diamandis,1,2* George M. Yousef,1,2 Judith Clements,3 Linda K. Ashworth,4 Shigetaka Yoshida,5 Torbjorn Egelrud,6 Peter S. Nelson,7 Sadao Shiosaka,5 Sheila Little,8 Hans Lilja,9 Ulf-Hakan Stenman,10 Harry G. Rittenhouse,11 and Hester Wain12 (1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada; 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario M5G 1L5, Canada; 3 Centre for Molecular Biotechnology, School of Life Sciences, Queensland University of Technology, Brisbane, Australia 4001; 4 Human Genome Center, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551; 5 Division of Structural Cell Biology, Nara Institute of Science and Technology, 8916-5 Takayama Ikoma, Nara 630-0101, Japan; 6 Department of Dermatology, University Hospital, S-901 85 Umeå, Sweden; 7 Department of Molecular Biotechnology, University of Washington, Seattle, WA 98105; 8 Central Nervous System Research, Lilly Research Laboratories, Indianapolis, IN 46285; 9 Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, S-20502 Malmo¨, Sweden; 10 Department of Clinical Chemistry, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland; 11 Hybritech Inc., PO Box 269006, San Diego, CA 92196; 12 Human Gene Nomenclature Committee, The Galton Laboratory, University College, London NW1 2HE, United Kingdom, * address correspondence to this author at: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario M5G 1X5, Canada; e-mail ediamandis@ mtsinai.on.ca)