diagnostic single nucleotide polymorphism analysis of factor v leiden and prothrombin 20210g>a

Coagulation and Transfusion Medicine / SNPS BY NANOCHIP, LIGHTCYCLER, AND RFLP

490 Am J Clin Pathol 2003;119:490-496490 DOI: 10.1309/3VTR7TL2X7TXL0QY

© American Society for Clinical Pathology

Diagnostic Single Nucleotide Polymorphism Analysis of Factor V Leiden and Prothrombin 20210G>A

A Comparison of the Nanogen Electronic Microarray With Restriction Enzyme Digestion and the Roche LightCycler

Iris Schrijver, MD,1,2 Marla J. Lay,2 and James L. Zehnder, MD1,2

Key Words: Thrombophilia; Factor V; Prothrombin; Factor II; Microarray; LightCycler; NanoChip; Restriction fragment length polymorphism; RFLP

DOI: 10.1309/3VTR7TL2X7TXL0QY

A b s t r a c t

Genetic thrombosis risk factors include a sequencevariant in the prothrombin gene (20210G>A) andfactor V Leiden (1691G>A). These single nucleotidepolymorphisms can be diagnosed with restrictionfragment length polymorphism (RFLP) analysis,fluorescent genotyping on the LightCycler (RocheDiagnostics, Indianapolis, IN), and microarray-basedtesting on the novel NanoChip electronic microarray(NanoChip Molecular Biology Workstation, Nanogen,San Diego, CA). We compared these methods foraccuracy, time to results, throughput, andinterpretation. Results from 789 of 800 individualamplicons analyzed on the NanoChip were in completeagreement with the other assays. Eleven were “nocalls” (uninterpreted by the NanoChip system) resultingfrom failed polymerase chain reaction amplifications.Although the NanoChip System, when used in a low-throughput setting, requires more overall time than theLightCycler, it is nearly equivalent per genotyping call.Owing to minimal sample handling, assay results aremore reliable on the NanoChip platform and on theLightCycler than with RFLP. The NanoChip assay isreliable and may be especially valuable to laboratorieswith a large volume of thrombophilia test requests.

Thromboembolic events have a combined frequency of 1to 2 per 1,000 individuals per year and demonstrate anincreased incidence with age.1 Mutations in genes that encodeblood coagulation factors can predispose to thrombotic disor-ders. A major inherited risk factor is mutation 1691G>A inthe factor V gene on chromosome 1q23 (factor V Leiden).This mutation results in resistance to factor Va cleavage byactivated protein C2,3 and results in persistence of the activestate, increased thrombin synthesis, and higher levels ofprothrombin fragment 1.2.4,5 The mutant protein confers a 5-to 10-fold risk of deep venous thrombosis (DVT) in heterozy-gous individuals and a 50- to 100-fold risk in homozygotes.6

Factor V Leiden is identified in approximately 20% ofpatients diagnosed with venous thromboembolism.7 Anotherrisk factor, associated with a 3-fold increased risk of DVT, issequence variant 20210G>A in the 3'-UTR (untranslatedregion) of the prothrombin gene on chromosome 11. Thegeneral carrier frequency of this mutation is 1% to 3% butapproaches 6% to 18% in patients with DVT.7 For reasonsthat remain obscure, factor V Leiden and prothrombin20210G>A are coinherited more often than expected.8

Genetic analysis of factor V Leiden may be used in cases withequivocal results by a modified activated protein C resistancescreening assay9 or to differentiate heterozygosity fromhomozygosity to provide appropriate clinical management.10

In addition, DNA analysis for factor V Leiden and other DVTrisk factors including prothrombin 20210G>A is increasinglyperformed routinely in conjunction with coagulation assays todefinitively determine the underlying risk factors for DVT.

To evaluate a newly developed electronic microarrayassay (Nanogen, San Diego, CA) for factor V Leiden andprothrombin 20210G>A, we performed a comparison for

Coagulation and Transfusion Medicine / ORIGINAL ARTICLE

Am J Clin Pathol 2003;119:490-496 491491 DOI: 10.1309/3VTR7TL2X7TXL0QY 491


accuracy, assay duration, hands-on time, and performancecomplexity with 2 diagnostic assays used in the molecularpathology laboratory. The first, a multiplexed polymerasechain reaction (PCR), followed by restriction digestion andpolyacrylamide gel electrophoresis, was used before imple-mentation of separate factor V Leiden and prothrombin20210G>A assays on the Roche LightCycler (Roche Diag-nostics, Indianapolis, IN), our current method.11,12 TheLightCycler assay enables amplification of genomic DNA inthin borosilicate glass capillaries under very rapid cyclingconditions. During PCR, the number of amplicons generatedis measured directly by increasing fluorescence, which iscreated by fluorescence resonance energy transfer from onelabeled probe to another. The 2 probes, one of which over-laps the interrogated single nucleotide polymorphism (SNP),are hybridized to neighboring areas of the amplicon duringPCR. After the amplification reaction, slowly increasingtemperatures cause dissociation of the hybridized probes andsubsequent reduction in the fluorescent signal. This providesa melting curve, which demonstrates earlier dissociationowing to a nonperfect match in the presence of themutation.13 The dissociation temperatures are sequence-specific and highly reproducible, so that wild-type samplescan be distinguished readily from heterozygous or homozy-gous sequences. The NanoChip Molecular Biology Worksta-tion (Nanogen) consists of a loader that electronicallyaddresses a previously amplified biotinylated PCR product tospecified locations on the NanoChip array. Subsequently,wild-type and mutant reporter oligonucleotides with differentfluorescent labels (Cy3 and Cy5, respectively) are passivelyhybridized to the NanoChip array. The hybridization isstrengthened with stabilizer oligonucleotides. Detection offluorescent signals occurs on the reader module, andcomputerized results are displayed in histograms, tables, andcharts. Wild-type, homozygous, and heterozygous samplesare identified based on relative red-green fluorescence.14

Materials and Methods

Samples

Four hundred individual patients who were tested previ-ously for the presence of factor V Leiden (1691G>A) andprothrombin gene variant 20210G>A in the Stanford Univer-sity Medical Center Molecular Pathology Laboratory, Stan-ford, CA, were included in the study. Samples from 200 ofthese patients were analyzed initially by restriction enzymeanalysis of multiplexed factor V–prothrombin PCR ampli-cons. DNA was extracted from peripheral blood leukocytesusing the Puregene DNA purification kit (Gentra Systems,Minneapolis, MN) or the Qiagen spin column (Qiagen,

Valencia, CA). Samples from the other 200 patients wereassayed using the LightCycler instrument. Blood samplesthat were assayed on the LightCycler were extracted on theMagnaPure instrument according to the manufacturer’srecommendations (Roche Diagnostics).

PCR Amplification and Restriction Enzyme Analysis

A 267-base-pair (bp) PCR product, containing a 121-bpsegment of factor V exon 10 and a fragment of intron 10,was generated with a primer pair, originally described byBertina et al,2 that flanks the factor V Leiden mutation. Theprimers for amplification of a part of exon 14 and the partial3'-UTR of the prothrombin gene were derived from Poort etal15 and encompass the prothrombin sequence variant20210G>A. The factor V and prothrombin fragments wereamplified in a multiplexed PCR reaction with AmpliTaqGold DNA polymerase, 250 U, 5 U/µL (Applied Biosystems,Foster City, CA) on a 9600 or 9700 Perkin Elmer thermocy-cler (Perkin Elmer, Shelton, CT). PCR cycling conditionscomprised a touchdown procedure16 and included 1 cycle at95°C for 10 minutes, 20 cycles at 95°C for 30 seconds, 62°Cfor 30 seconds with a 1°C decrease in temperature per cycle,and 72°C for 30 seconds. This was followed by 15 cycles of95°C for 30 seconds, 42°C for 30 seconds, and 72°C for 30seconds. There was a 5-minute extension at 72°C. After PCRamplification, 5 U of restriction enzyme MnlI and 20 U ofHindIII (New England Biolabs, Beverly, MA) were added toeach PCR tube.17 Following a 60-minute incubation at 37°C,2 µL of each sample was electrophoresed for 60 minutes on a10% polyacrylamide gel at 200 V. The gels were stainedwith the GelStar staining solution (BioWhittaker, Walk-ersville, MD), and bands were photographed under UV illu-mination. The expected size for a homozygous wild-typeprothrombin amplicon is 345 bp, whereas the homozygous20210G>A is cut by HindIII and expected at 322 and 23 bp.Restriction analysis of the wild-type factor V fragment yieldswild-type bands of 163, 67, and 37 bp. Factor V Leiden,however, abandons 1 of the 2 restriction sites, resulting infragments of 200 and 67 bp18 ❚ Image 1A❚ .

LightCycler Amplification

The LightCycler–factor V Leiden and the LightCycler-prothrombin mutation detection sets (Roche) were used inconjunction with the MagnaPure instrument to preparesamples for subsequent amplification on the LightCyclerinstrument according to the manufacturer’s guidelines. Inbrief, approximately 250 ng of patient DNA was added to a5-µL PCR mix provided in the set, which, among otherproprietary reagents, contains the forward and reverseprimers for either the factor V or the prothrombin amplifica-tion reaction and fluorescently labeled donor and acceptorhybridization probes. The LightCycler conditions for both

Schrijver et al / SNPS BY NANOCHIP, LIGHTCYCLER, AND RFLP



assays are identical. The samples were denatured at 95°C for10 minutes, followed by 40 cycles of 95°C for 15 seconds,55°C for 10 seconds, and 72°C for 10 seconds. This wasfollowed by 1 melting curve cycle, which begins with anincrease in temperature to 95°C to denature the amplicons,followed by a 30-second pause at 40°C and a slow increaseat a temperature rate of 0.1°C per second to 80°C. The finalcooling cycle is at 40°C for 30 seconds. The cycles arefollowed by a display on the computer screen of the fluores-cence intensity vs temperature and derived melting curves❚ Image 1B❚ . The typical temperature for prothrombin20210G is 58°C, and for 20210A it is 49°C. The probemelting temperature for wild-type factor V is 65°C, whereasfactor V Leiden it is 57°C.

PCR Amplification and NanoChip Platform Analysis

The extracted DNA, which previously was used with therestriction enzyme assay or the LightCycler analysis, now wasamplified with the Nanogen primer sets for multiplexed ampli-fication of the factor V and prothrombin fragments thatencompass the SNPs of interest. PCR amplification withforward primers and biotinylated reverse primers wasperformed using AmpliTaq Gold DNA polymerase 250 U, 5U/µL, and the four 2'-deoxynucleotide 5'-triphosphates (Amer-sham Pharmacia, Piscataway, NJ) on a 9600 Perkin Elmerinstrument. Amplification conditions were as follows: 95°Cfor 10 minutes, 40 cycles of 94°C for 20 seconds, 55°C for 20seconds, and 72°C for 45 seconds, and a final extension of72°C for 5 minutes. Following amplification, the PCR sampleswere desalted on a Millipore Multi Screen 96-well desaltingplate (Millipore, Bedford, MA) and mixed with a 50-mmol/Lconcentration of histidine. A volume of 60 µL of each samplewas transferred into a Nunc 96-well plate (Nalge Nunc,Rochester, NY) and prepared for the NanoChip loaderaccording to the manufacturer’s guidelines. The samples wereaddressed electronically to pads on the NanoChip cartridge.Each sample was run in duplicate and placed on adjacent pads.

On completion of the loader map file, the cartridgeswere rinsed with a high-salt buffer (50-mmol/L concentrationof sodium phosphate, pH 7.4, 500-mmol/L concentration ofsodium chloride). Two reporter-stabilizer mixes for the factorV and prothrombin assays were prepared as specified in themanufacturer’s protocol. First, the factor V mix was pipettedonto the microarray and incubated for 3 minutes, followed bya wash with the high-salt buffer. Next, the cartridge wasplaced onto the NanoChip reader instrument and analyzed atthe factor V Leiden discrimination temperature (31°C). Thehybridized factor V Leiden probe was removed from thecartridge by a series of washes with 0.1N NaOH, deionizedH2O and high-salt buffer. The procedure was repeated usingthe prothrombin reporter mix. Results were interpreted at adiscrimination temperature of 36°C ❚ Image 1C❚ .

Results

Accuracy

A novel diagnostic system for identification of factor VLeiden 1691G>A and prothrombin 20210G>A wascompared with restriction enzyme digestion and the Light-Cycler assays. In our comparison with the 200 samples origi-nally genotyped by restriction enzyme analysis, results wereobtained in 197 of 200 samples for factor V and 199 of 200for prothrombin. All obtained results were identical betweenthe assays. Failure of amplification for both factor V andprothrombin occurred in a single sample and may have beenbecause the DNA was of low concentration and several yearsold. Testing of this sample could not be repeated because noDNA was left after the original comparison. Whereas theprothrombin segment in the multiplex PCR reaction wasamplified, 2 samples failed factor V amplification, evenwhen a separate factor V primer set was used.

In our comparison with the 200 samples originally geno-typed by the LightCycler, results were obtained in 195 of 200samples for factor V and 198 of 200 for prothrombin. Allresults obtained on the NanoChip platform were entirelycongruent with the LightCycler results. To assess whetherfailure was run-specific or due to sample compromise resultingfrom DNA degradation or the presence of PCR inhibitors, weattempted reamplification on both the NanoChip System andon the LightCycler of 5 samples that initially failed using theNanogen primers. Of these 5 samples, testing on 2 could notbe repeated because no sample was left, and 3 were amplifiedsuccessfully only with the LightCycler primer set. In 1 resulton the NanoChip System, a nontemplate control sampleproduced an indeterminate result. Of the 2 sample pads, thepad immediately next to the positive sample was weakly posi-tive, whereas the second pad remained negative. When thesample was tested again, it gave a negative result.

Assay Duration and Throughput

The time requirement for DNA extraction was similar forall 3 assays. The restriction fragment length polymorphism(RFLP) approach requires a PCR setup in which we use 28patient samples, a control heterozygous for both mutations ofinterest, and a control without template. After the PCR ampli-fication, restriction enzymes are added to each tube. Thesamples are incubated at 37°C for 1 hour. During this time, 30minutes are used for the preparation of 4 polyacrylamide gels❚ Figure 1❚ ❚ Table 1❚ . The samples are loaded onto the gels andelectrophoresed, followed by interpretation of the results.

The LightCycler analysis is preceded by a PCR prepara-tion for 32 samples. This is the maximum number of samplesthat can be analyzed per run on a single Light-Cycler instru-ment (Figure 1, Table 1). We include a DNA negative controland a factor V Leiden and/or a prothrombin 20210G>A




heterozygous control in each analysis. After computerprogramming of the MagnaPure instrument, the MagnaPureloads the PCR reagents and patient DNA into the appropriatecapillaries in about 35 minutes. The samples undergo a

temperature cycle program on the LightCycler, and the resultsare interpreted directly after completion.

The Nanogen assay begins with sample preparation and amultiplexed PCR reaction. The amplicons are desalted on a 96-well plate and transferred to a Nanogen loader plate, after whichuser-defined computer sample-map files are created and controlsprepared. The sample plate and the cartridges are put into theloader and processed for 270 minutes (throughput depends on thenumber of samples being tested). During this time, the samples areaddressed electronically to their designated positions on theNanoChip array. The subsequent hybridization with the factor Vprobe occurs outside the loader, after which the cartridge ismoved into the reader, scanned, and interpreted by the Nanogensoftware. The hybridization and reader steps are repeated withthe prothrombin probe. Loader flush and shutdown arecompleted during interpretation of the results (Figure 1, Table 1).It should be noted that Nanogen claims 96 samples can be loadedin 295 minutes owing to parallel fluidic processing. However,we did not validate this by testing 96 samples at one time.

In our comparison, we tested only 48 samples (96 calls)on 1 cartridge, although the Nanogen loader can hold up to 4

1 2 3 4 5 6 7 8 9 10

345 bp322 bp

200 bp

163 bp

❚ Image 1❚ A, Restriction fragment length polymorphismanalysis. The products of the factor V Leiden/prothrombin20210G>A multiplex polymerase chain reaction (PCR) reactionare visualized on polyacrylamide gel electrophoresis afterrestriction digestion. Bands under 100 base pairs (bp) are notshown. Lane 1, 100-bp DNA ladder; lanes 2-5, normal factor V(163 bp) and prothrombin (345 bp); lanes 6 and 7, homozygouswild-type for factor V Leiden (163 bp) and heterozygous for20210G>A (345- and 322-bp bands due to digestion withHindIII); lane 8, homozygous for 20210G (345 bp) andheterozygous for factor V Leiden (163 and 200 bp afterdigestion with MnlI); lane 9, control sample, heterozygous forfactor V Leiden and prothrombin 20210G>A; lane 10, negativecontrol. B, LightCycler analysis. Melting profile of a samplethat is homozygous wild-type for factor V Leiden (1691G,green), superimposed on a heterozygous control (1691G>A,blue). The typical melting temperature for the wild-typesequence is 65°C, but in the presence of factor V Leiden,melting occurs at 57°C. C, NanoChip display. One mode ofdata presentation in the NanoChip prothrombin 20210G>Aassay. Heterozygous samples are made up of half red and halfgreen circles, whereas homozygous mutant samples are redonly and homozygous wild-type samples are green only. Thisdisplay is an approximation of the software analysis, which ismore precisely summarized in detailed tables and graphs.Differences in signal strength most likely reflect variation inPCR efficiency. Ref, positive heterozygous prothrombincontrol, supplied by the manufacturer; Pt, patient who isheterozygous for prothrombin 20210G>A and homozygous forfactor V Leiden. For proprietary information, see the text.

Ref

Pt

A

B

C




cartridges. Each electronic microarray has 100 locations andcan be addressed singly, but we addressed each sample induplicate for quality control purposes (Figure 1, Table 1).With each run, we included 1 sample without template andheterozygous reference controls for factor V Leiden andprothrombin 20210G>A. These were provided by the manu-facturer. In addition, we chose to include 1 known patient

control sample, which was homozygous for factor V Leidenand heterozygous for the prothrombin mutation.

Hands-on Time

The time of actual involvement by the molecular tech-nologist is 1 hour and 40 minutes for the RFLP assay for 30samples (60 genotyping calls) and 1 hour and 20 minutes forLightCycler analysis for 32 samples (32 genotyping calls)(Figure 1, Table 1). Per sample, this translates to 3.3 minutesfor the restriction enzyme assay and 2.5 minutes for theLightCycler. In the NanoChip System evaluation, 2 hoursand 35 minutes of technologist time were required forcompletion of the assay. Hands-on time for this assay is 3.2minutes per sample because we used 48 samples (96 geno-typing calls) per Nanochip cartridge and 2 electronic padsper sample. The hands-on time per genotyping call translatesto 1.7 minutes for the RFLP assay, 2.5 minutes for the Light-Cycler, and 1.6 minutes for the NanoChip System.

Discussion

Microarrays can be manufactured by directly generatingnucleic acid probes on the solid support, by straight transfer

0 1 2 3 4 5 6 7 8 9 10 11

RFLP

LightCycler

NanoChip

Hours

❚ Figure 1❚ Comparison of total assay time and hands-on timefor restriction fragment length polymorphism (RFLP) analysis,LightCycler amplification, and the NanoChip assay. The time,displayed in hours, indicates the duration of each assay.Shaded boxes represent hands-on-time, and clear boxesrepresent the time during which the assay continues withoutdirect involvement. For proprietary information, see the text.

❚ Table 1❚Time Study Details*

Restriction Fragment NanogenLength Polymorphism Roche LightCycler Electronic Microarray

Specimen preparation, minPCR 30 40 30MagnaPure programming — 25 —MagnaPure loading — 35 —

Procedure, minPCR amplification 90 65 132Enzyme addition 10 — —Incubation 60 — —Gel loading 15 — —Electrophoresis 60 — —Desalting — — 45Sample map creation — — 35Loader — — 270Hybridization with factor V probe — — 10Reader — — 20Stripping of the microarray — — 10Hybridization with prothrombin probe — — 10Reader — — 20

Results, minInterpretation 15 15 15

Time and throughputTime to results 4 h 40 min 3 h 9 h 57 minHands-on time 1 h 40 min 1 h 20 min 2 h 35 minThroughput used in the present study 30 32 48Genotyping calls 60 32 96Overall time per sample, min 9.3 5.6 12.4Overall time per genotyping call, min 4.7 5.6 6.2Hands-on time per sample, min 3.3 2.5 3.2Hands-on time per genotyping call, min 1.7 2.5 1.6Maximum throughput per run 30 32 192 (384:2)

PCR, polymerase chain reaction.* For proprietary information, see the text.




of prefabricated probes onto the array, or by electronic mobi-lization of oligonucleotides to pads on an active microelec-tronic chip.19 In an electronic dot-blot method developed byNanogen, the use of electric fields and an L-histidine bufferwith minimal conductivity permits rapid transport and elec-tric field concentration of DNA to defined locations on thearray, as well as optimized hybridization.19 Hybridizationstringency is achieved by reversal of polarity at optimizedelectric field strength, which repulses the charged moleculesthat lack optimal affinity for the substrate.20 A porous strep-tavidin-containing permeation layer covers the microelec-trodes and facilitates interaction with small ions and waterbut protects larger molecules such as DNA from damage inthe semiconductor microchip.21 Since our evaluation of theNanoChip System, the permeation layer has been modifiedfrom an agarose-based to a non–protein-based hydrogelmatrix. The NanoChip Molecular Biology Workstationincludes a loader, which electronically addresses the biotiny-lated PCR amplicon to specified locations on the microchip.Subsequently, a stabilizer oligonucleotide and wild-type andmutant reporter oligonucleotides with different fluorescentlabels (Cy3 and Cy5, respectively) are passively hybridizedto the chip. Detection of fluorescent signals occurs on thereader module, and computerized results are displayed inhistograms, tables, and charts.14

Of the 200 RFLP samples that were compared in ourNanogen evaluation, 19 were heterozygous for factor VLeiden and 2 were homozygous for this mutation. Nineteensamples were heterozygous for prothrombin 20210G>A, and1 was homozygous. Factor V Leiden NanoChip results couldbe obtained for 197 of 200 patient samples. These were iden-tical to the RFLP findings, as were the 199 of 200 results forprothrombin 20210G>A. Results for 200 patient sampleswere compared with results on the Roche LightCycler. Forthis assay, we included 20 heterozygous and 2 homozygousfactor V Leiden samples. The positive prothrombin20210G>A samples included 1 homozygous and 19heterozygous specimens. With the NanoChip, we obtained195 of 200 factor V Leiden and 198 of 200 prothrombin20210G>A results. Three samples that could not be ampli-fied with the Nanogen primers in a nonmultiplexed reactionwere reamplified on the LightCycler to assess sampleintegrity. Amplification was achieved, indicating that theLightCycler amplification may be better optimized.

Two samples, from a woman and her newborn child,displayed an atypical melting curve pattern on LightCycleranalysis ❚ Image 2❚ . The LightCycler assay will detect othersequence alterations under the mutation probe, with alteredmelting patterns observed. To confirm that the underlyingmutation was not at nucleotide position 20210, the sampleswere sequenced directly and were called as homozygous wild-type for prothrombin at position 20210 (data not shown).

However, both samples were heterozygous for prothrombinsequence variants near the SNP of interest. The variants inthese samples may represent innocent polymorphisms.Despite the proximity of these variants to the SNP ofinterest, the NanoChip assay correctly identified these 2samples as homozygous wild-type for prothrombin20210G>A, indicating that the NanoChip System may notbe affected by variants of this type.

The results from the 400 multiplexed amplicons (800genotyping calls) analyzed on the NanoChip System resultedin overall 2.0% and 0.8% no-call rates for factor V andprothrombin, respectively. Eight samples contributed to theno-call rate due to failed PCR: 3 samples failed PCR forboth factor V and prothrombin and 5 samples failed PCR forfactor V only. There was 1 instance of an indeterminatenontemplate control with the NanoChip assay. A single posi-tive pad was flanked by a strongly positive patient controlsample on the previously addressed pad and by the second(negative) nontemplate pad on the other side. By running induplicate, the discrepancy was identified readily. The DNAconcentration of the positive patient control adjacent to theindeterminate nontemplate control sample was 100 ng/µL.As with all other PCRs in this assay, 200 ng of DNA wasused in the amplification reaction. The negative control

❚ Image 2❚ LightCycler analysis. The melting profile of aheterozygous control (20210G>A, blue) reveals 1 peak at thetypical melting temperature for the wild-type sequence(58°C) and 1 peak that corresponds to presence of themutation, which decreases the melting temperature to 49°C.The atypical melting curve for 1 patient sample (green) issuperimposed onto this curve and demonstrates 1 wild-typemelting temperature (58°C) and 1 unknown allele at 53°C.Direct DNA sequencing confirmed that the patient ishomozygous for the wild-type sequence, prothrombin20210G. For proprietary information, see the text.




sample was run on a polyacrylamide gel to rule out possiblecontamination during the PCR reaction. As no PCR productwas visualized, the same sample was addressed onto anotherchip and subsequently read as negative. More extensive eval-uations have shown no evidence of cross-contaminationbetween test sites.22 However, performing sample testing induplicate initially, to verify the absence of cross-contamina-tion, may be indicated for quality assurance.

The NanoChip technology of electronic assignment ofDNA sequences to pads on a microarray has been applied tothe diagnosis of factor V Leiden and prothrombin20210G>A. After a brief training period, the assay is rela-tively easy to perform and accurate. Other advantagesinclude the possibility of very high throughput, extensiveautomation with minimal risk of sample error, and compre-hensive results display on the computer screen after comple-tion of the assay. One disadvantage may include a longertime to results compared with the LightCycler assay if theinstrument is not used in a high-throughput setting. TheNanoChip assay may find application in smaller laboratoriesonce the assay is automated further or completed in a shortertime. Currently, it may be of special interest for moleculardiagnostic laboratories with a medium to large test volume.

From the 1Department of Pathology and the 2Molecular PathologyLaboratory, Stanford University Medical Center, Stanford, CA.

Supported in part by Nanogen.Address reprint requests to Dr Schrijver: Dept of Pathology,

L235, Stanford University Medical Center, Stanford, CA 94305.Acknowledgment: We thank Carol D. Jones for technical

assistance.

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diagnostic single nucleotide polymorphism analysis of factor v leiden and prothrombin 20210g>a

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