TY - JOUR
AU - Knight, Ivor, T
AB - Abstract BACKGROUND Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.*97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platform's liquid-handling system automated PCR and HSM. RESULTS PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS We demonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis. Most genetic diseases are rare. Laboratories typically lack technology to offer rapid testing for rare diseases or genotypes. Individual samples are seldom processed. Instead, multiple samples are collected for batch analysis, often resulting in slow turnaround times varying from a few days to several weeks. Despite the extraordinary amount of information collected by massively parallel sequencing, the focus is not on turnaround time, and costs remain high for single-gene analysis compared with scanning by high-resolution melting (1). Turnaround times are often slow in PCR-based genetic tests because multiple reactions are performed in parallel by use of programmable thermal cyclers. These methods are typically restricted to a single protocol, placing constraints on assay design. A DNA sample may need to be processed multiple times to accommodate varying protocols for a panel of tests. As a result, consumables, turnaround times, and laboratory errors all increase, thus decreasing throughput. In addition, some protocols require additional reflex testing based on the initial result outcome. This typically requires more processing that may require additional batching and delay. Some molecular tests with a limited number of targets use single-use individual cartridges. A few can be rapidly performed, but at a cost that may not be acceptable to the laboratory. Fast molecular testing usually requires specialized sample containers. For example, rapid-cycle PCR and high-resolution melting can be performed quickly in capillaries (2), but individual capillaries are difficult to handle in high numbers. We have developed an integrated microfluidic platform with serial, rapid-cycle PCR and high-speed melting (HSM)5 analysis. Sequential discrete reactions within microfluidic channels allow variable thermal cycling and melting parameters for each reaction. The volume scale-down of microfluidics enables rapid heat transfer for faster thermal cycling (3) as well as rapid melting rates for HSM. HSM, a faster variant of high-resolution melting, provides a simple method to analyze PCR products by use of a saturating fluorescent dye to detect sequence variation. HSM analysis minimizes time and cost after PCR (1), allowing genotyping (4), variant scanning (5), and other applications (6, 7). In the present study, we demonstrate the genotyping accuracy of a custom prototype instrument that analyzes up to 8 samples in parallel by small amplicon genotyping (8) and HSM. Materials and Methods CLINICAL SAMPLES We obtained previously genotyped and deidentified (University of Utah Institutional Review Board #7275) blood samples from Associated Regional and University Pathologists (ARUP) Laboratories. After enrichment for rare F5 [coagulation factor V (proaccelerin, labile factor)] homozygotes,6F2 [coagulation factor II (thrombin)] heterozygotes, and F2 homozygotes, DNA was extracted (Gentra Puregene Blood Kit, Qiagen) and diluted to 200 ng/μL with 10 mmol/L Tris, pH 8.0, 0.1 mmol/L EDTA with A260 (Nanodrop 1000, Thermo Scientific). We selected 105 samples with an A260/A280 ratio between 1.7 and 2.0, genotyped them by high-resolution melting assays on commercial instruments, and used ≥1 of the following: small amplicon genotyping (8), unlabeled probe genotyping (9), or snapback primer genotyping (10). Samples were then blinded into two 100-sample lots, with 1 lot tested at Canon US Life Sciences (Rockville, MD) and the other at the University of Utah (Salt Lake City, UT). DNA CONTROLS AND OLIGONUCLEOTIDES We designed primers for 4 small amplicon genotyping assays to detect common single nucleotide variants associated with thrombophilia: F2 c.*97G>A (11), F5 c.1601G>A (12), and MTHFR [methylenetetrahydrofolate reductase (NAD(P)H)] c.665C>T (13) and c.1286A>C (14). An internal temperature control of complementary oligonucleotides was included in each assay for better temperature precision (15). An ultraconserved element (16) on chromosome 17 was also amplified and melted in all channels 1 time before the panel of 4 loci in this study. It was used as an additional control during instrument development but is not required for future system use. Control DNA, wild-type at F2, F5, and the MTHFR loci, was extracted from the GM11254 cell line (Coriell) by column absorption (Blood and Cell Culture DNA Kit, Qiagen). We designed primers for high-temperature (90.0 °C) and low-temperature (69.9 °C) calibration of the instrument to amplify artificial plasmid sequences not analogous to the human genome. Primers, controls, and calibrator sequences were synthesized, HPLC purified, and resuspended in 10 mmol/L Tris, 0.1 mmol/L EDTA at pH 8.0 (Integrated DNA Technologies). DNA duplex length, guanine-cytosine content, and measured melting temperature (Tm) values are shown in Table 1, and primer and control sequences are given in Supplemental Table 1, which accompanies the online version of this article at http://www.clinchem.org/content/vol60/issue10. DNA duplex length, guanine-cytosine (GC) content, and measured Tm values. Table 1. DNA duplex length, guanine-cytosine (GC) content, and measured Tm values. DNA duplex . Length, bp . GC content, % . Tm, °Ca . ΔTm, °Cb . Ultraconserved sequence 81 44 80.5 F2 c.*97G>A 48 50 78.8 0.8 F5 c.1601G>A 43 49 76.6 0.8 MTHFR c.665C>T 48 52 79.6 0.9 MTHFR c.1286A>C 46 46 76.6 −1.1 Internal temperature control 45 71 85.0 Low Tm calibrator 100 15 69.9 High Tm calibrator 200 61 90.0 DNA duplex . Length, bp . GC content, % . Tm, °Ca . ΔTm, °Cb . Ultraconserved sequence 81 44 80.5 F2 c.*97G>A 48 50 78.8 0.8 F5 c.1601G>A 43 49 76.6 0.8 MTHFR c.665C>T 48 52 79.6 0.9 MTHFR c.1286A>C 46 46 76.6 −1.1 Internal temperature control 45 71 85.0 Low Tm calibrator 100 15 69.9 High Tm calibrator 200 61 90.0 a Measured Tm of the wild-type sequence. b Tm of the wild-type minus the Tm of the homozygous variant. Open in new tab Table 1. DNA duplex length, guanine-cytosine (GC) content, and measured Tm values. DNA duplex . Length, bp . GC content, % . Tm, °Ca . ΔTm, °Cb . Ultraconserved sequence 81 44 80.5 F2 c.*97G>A 48 50 78.8 0.8 F5 c.1601G>A 43 49 76.6 0.8 MTHFR c.665C>T 48 52 79.6 0.9 MTHFR c.1286A>C 46 46 76.6 −1.1 Internal temperature control 45 71 85.0 Low Tm calibrator 100 15 69.9 High Tm calibrator 200 61 90.0 DNA duplex . Length, bp . GC content, % . Tm, °Ca . ΔTm, °Cb . Ultraconserved sequence 81 44 80.5 F2 c.*97G>A 48 50 78.8 0.8 F5 c.1601G>A 43 49 76.6 0.8 MTHFR c.665C>T 48 52 79.6 0.9 MTHFR c.1286A>C 46 46 76.6 −1.1 Internal temperature control 45 71 85.0 Low Tm calibrator 100 15 69.9 High Tm calibrator 200 61 90.0 a Measured Tm of the wild-type sequence. b Tm of the wild-type minus the Tm of the homozygous variant. Open in new tab RAPID-CYCLE PCR AND HSM ANALYSIS PCR was performed within microchannels that contained approximately 50-nL reaction volumes. The reactions included 1× Titanium® Taq DNA Polymerase (Clontech), 3.0 mmol/L MgCl2, 50 mmol/L Tris-HCl (pH 8.0), 0.04% Tween® 20 (Thermo Fisher Scientific), 1.0 mol/L betaine monohydrate (Sigma-Aldrich), 2% DMSO, 50 mmol/L KCl, 0.2% BSA, 375 μmol/L of each dNTP, 1× LCGreen® Plus (BioFire Diagnostics), 1.0 μmol/L of each test primer, 0.5 μmol/L internal temperature duplex, and 18 ng/μL human genomic DNA. The ultraconserved element PCR did not include the internal temperature control duplex. The PCR protocol included an initial denaturation step of 95 °C for 30 s followed by 40 cycles of 95 °C for 1.5 s (50 °C/s ramp rate), 60 °C for 2.5 s (20 °C/s ramp rate), and 72 °C for 2.5 s (2 °C/s ramp rate). After PCR, a denature/renature step was implemented at 95 °C for 1.5 s (50 °C/s ramp rate) and 50 °C for 2 s (20 °C/s ramp rate) before melting the product. The product was then melted from 65 °C to 95 °C with a 0.5 °C/s ramping rate to produce HSM curves, thus providing a temperature resolution of approximately 0.02 °C. Melting curves were interpreted with custom high-resolution melting software (17). Briefly, the fluorescence melting curve data was first extracted by removing the exponential background. Then, the negative derivative of the melting curve was obtained by differentiating the second-degree polynomial best fit with Savitzky–Golay moving windows (18). The temperature control peak maximum (Tm) was used to overlay each curve so that the control Tm was translated to the mean control Tm of all curves. Homozygous samples were differentiated by Tm, by use of knowledge of the measured Tm difference outlined in Table 1 and the wild-type control as a reference point along with the aid of clustering software (19) to help define wild-type and homozygous variant groupings. Heterozygous samples were distinguished by shape differences, caused by the presence of heteroduplexes. MICROFLUIDIC PLATFORM AND WORK FLOW Before starting the instrument run, DNA samples were mixed with the PCR reagents and placed on the 384-well plate, as indicated in online Supplemental Fig. 1. The calibrator was also placed on the plate as indicated, and the blanking solution was pipetted onto the cartridge. The cartridge has 2 components, a “world-to-chip” poly(methylmethacrylate) plastic interface that surrounds a glass chip component where PCR and HSM are performed (Fig. 1). These consumables were placed in the instrument drawer together with a tip station that was used to house the pipette tips and foam rollers that removed excess fluid from the pipette tips in between fluid deliveries (Fig. 2). (A), Photograph of the fully assembled 8-channel microfluidic cartridge containing the core glass chip; (B), Schematic of the glass chip labeled with primary components. Fig. 1. Open in new tabDownload slide Fig. 1. Open in new tabDownload slide Prototype instrument. Fig. 2. Open in new tabDownload slide (A), Schematic shows the liquid handling robots, a 384-reagent plate, a microfluidic cartridge, and an optical imaging system. The components are controlled and coordinated by custom LabVIEW software (National Instruments). The automated pipette system has 2 independent pipette heads that use linear actuators (Oriental Motor) to move custom pipette tips. One pipette head handles PCR reagents and moves between a 384-well microtiter plate (Greiner Bio-One), a tip station, and the microfluidic cartridge. The tip station contains custom pipette tips and a disposal area. The other pipette head handles a blanking solution, moving between different locations on the microfluidic cartridge. Syringe pumps with 50-μL capacity (TriContinent) are used for each pipette tip to aspirate and dispense fluids. Three microliters are aspirated from the 384-well microtiter plate, with 2 μL dispensed on the end of the tip forming a hemispherical droplet of reagent. This droplet is brought in contact with the capillary tubes on the cartridge to introduce the reagent to the microfluidic cartridge. (B), Photograph of the assembled prototype. (C), Photograph of the inside of the prototype. Inside the drawer from left to right are the microtiter plate, the tip station, and the microfluidic cartridge. Fig. 2. Open in new tabDownload slide (A), Schematic shows the liquid handling robots, a 384-reagent plate, a microfluidic cartridge, and an optical imaging system. The components are controlled and coordinated by custom LabVIEW software (National Instruments). The automated pipette system has 2 independent pipette heads that use linear actuators (Oriental Motor) to move custom pipette tips. One pipette head handles PCR reagents and moves between a 384-well microtiter plate (Greiner Bio-One), a tip station, and the microfluidic cartridge. The tip station contains custom pipette tips and a disposal area. The other pipette head handles a blanking solution, moving between different locations on the microfluidic cartridge. Syringe pumps with 50-μL capacity (TriContinent) are used for each pipette tip to aspirate and dispense fluids. Three microliters are aspirated from the 384-well microtiter plate, with 2 μL dispensed on the end of the tip forming a hemispherical droplet of reagent. This droplet is brought in contact with the capillary tubes on the cartridge to introduce the reagent to the microfluidic cartridge. (B), Photograph of the assembled prototype. (C), Photograph of the inside of the prototype. Inside the drawer from left to right are the microtiter plate, the tip station, and the microfluidic cartridge. Two fluid types, either the PCR reagents or the blanking solution, are delivered to 8 fused silica capillaries (Polymicro Technologies) assembled within the cartridge by use of 2 different liquid-handling robots. One robot and set of pipette tips is dedicated for PCR reagents, whereas the other delivers blanking solution. First, the blanking solution containing 35 μmol/L Alexa 647 fluorescent dye is delivered to the cartridge (see online Supplemental Fig. 2A). The solution moves into the cartridge and through the microchannels in the glass chip, where it is optically monitored via illumination with a red light-emitting diode (LED). Furthermore, the fluid motion is controlled by detecting the fluorescent edge of the blanking solution and giving feedback to the peristaltic pumps (Watson Marlowe) to control and confirm the solution position. Every other fluid delivery to the cartridge contains this blanking solution, whereas alternating deliveries contain the reagents for PCR/HSM (see online Supplemental Fig. 2). This delivery pattern forms the basis of a serial PCR stream where PCR reagents with specific primers are delivered to the cartridge in a sequential fashion. These specific reactions on the same DNA sample are separated by blanking solution. A maximum of 8 different samples are used in a run within the same cartridge (1 DNA sample/channel). Cartridge heater calibration is performed at the beginning of the run by melting a mixture of high- and low-Tm preamplified products as calibrators (Table 1). Then, the ultraconserved element PCR is (optionally) performed as a control. This is followed by multiple serial tests, where each locus to be genotyped is sequentially PCR amplified and melted. In our experimental setup, the 2 outer channels in each run were negative controls without genomic DNA, and 1 of the remaining 6 channels contained wild-type control DNA. Each of the microfluidic channels has individual heat control through thin-film platinum heaters embedded under each sample channel in the glass chip. The heaters are isolated from the fluid within the microchannel by a passivation layer of SiO2. The glass chip design includes gold metal traces that connect to flex circuits on the cartridge and interface with electrical components on the instrument to enable temperature control. Fans in the instrument move air across a heat sink attached to the glass chip to aid in the passive cooling of the platinum heaters during PCR. Additional technical specifications have been described (20). The optics to monitor fluid control and perform HSM were matched to LCGreen Plus and Alexa 647 fluorescent dyes. We used a 445-nm blue LED (Innovation In Optics), positioned 45° incident to the glass chip on the fluidic inlet side, with a 438DF24 excitation filter (Semrock) and a 629-nm red LED (Innovation In Optics), positioned 45° incident to the glass chip on the fluidic outlet side, with a 632DF22 excitation filter (Semrock) to illuminate the fluorescent dyes within the glass chip. Emitted fluorescent light was collected in an EF 50-mm F/1.4 USM lens (Canon U.S.A.), passed through a 510DF80/685DF50 dual bandpass filter (Semrock), and imaged, with an acquisition rate of 30 Hz for HSM and 1 Hz for other system operations, by use of a complementary metal–oxide semiconductor sensor (EOS 5D Mark II, Canon U.S.A.). Heaters used proportional/integral/derivative loop control with pulse width modulation. The thin-film platinum heaters in the microfluidic chip served as both heaters and temperature sensors. The microfluidic design combined with individual heating allows for rapid-cycle PCR (see online Supplemental Fig. 3). We used a PCR protocol with 40 cycles of 15 s each. This protocol, coupled with an HSM protocol with a 0.5 °C/s ramp rate from 65 °C to 95 °C, allowed a single PCR/HSM test to be completed in about 12.5 min, not including time for fluid delivery to the chip. Performing the panel of 5 PCRs used here (1 control and 4 genotyping loci) currently requires 2.5 h, including fluid delivery, system initialization, and calibration, steps that have not yet been optimized for speed. At the end of the run, the instrument generates a data output file that contains fluorescence vs temperature information. This file is then analyzed for visual genotyping (Fig. 3). Example melting curves for 6 samples at each locus clustered by genotype. Fig. 3. Open in new tabDownload slide The Human Genome Variation Society nomenclature for the variants are (A), F2 (c.*97G>A); (B), F5 (c.1601G>A); (C), MTHFR (c.665C>T); and (D), MTHFR c.1286A>C. The legacy names for these variants are F2 20210G>A, F5 1691G>A, MTHFR 677C>T, and MTHFR 1298A>C. The negative derivative melting plots are displayed after exponential background removal, normalization, and temperature overlay with the internal temperature control (ITC). The black curves are wild-type, the blue curves are homozygous variant, and the red curves are heterozygous variant. The y axis is the negative derivative of fluorescence with respect to temperature (–dF/dT). Fig. 3. Open in new tabDownload slide The Human Genome Variation Society nomenclature for the variants are (A), F2 (c.*97G>A); (B), F5 (c.1601G>A); (C), MTHFR (c.665C>T); and (D), MTHFR c.1286A>C. The legacy names for these variants are F2 20210G>A, F5 1691G>A, MTHFR 677C>T, and MTHFR 1298A>C. The negative derivative melting plots are displayed after exponential background removal, normalization, and temperature overlay with the internal temperature control (ITC). The black curves are wild-type, the blue curves are homozygous variant, and the red curves are heterozygous variant. The y axis is the negative derivative of fluorescence with respect to temperature (–dF/dT). Results The small amplicon genotyping products were 43–48 bp, with experimental Tm values of 76.6–80.5 °C (Table 1). The temperature difference between the 2 homozygous genotypes (ΔTm) at each locus varied from 0.8 to 1.1 °C as expected for class 1 and class 2 single base variants (8). The synthetic internal temperature control used to increase temperature precision melted 5.4 °C higher than any genotype at the 4 loci and did not interfere with genotyping. The mean Tm SD across channels decreased from 0.18 °C before temperature correction to 0.07 °C after temperature correction, allowing good separation of both homozygotes in all assays. The entire process (PCR, HSM, and melting curve display) required 12.5 min per assay. Representative melting curves, shown as their negative derivatives, are displayed in Fig. 3. As expected, different homozygotes were separated by Tm, and heterozygotes had a second low temperature peak consisting of heteroduplexes (8). An additional melting transition from the internal temperature control at 85.0 °C was used to overlay control Tm values across samples to increase temperature precision. The minimum difference in temperature between homozygous genotypes at any locus was 0.8 °C compared with a peak SD of 0.07 °C, enough separation that genotyping errors between homozygotes should be very rare. Genotyping accuracy was evaluated in 2 blinded studies. In each study, 100 samples were selected from the 105 available DNA samples and analysis was performed at either Canon US Life Sciences (Table 2) or the University of Utah (Table 3) for a total of 800 genotype analyses. In both blinded studies, no genotyping errors could be attributed to the instrument or HSM (100% accuracy). However, the melting curves of 6 assays (0.75%) were not definitive on the initial run and needed to be repeated once to verify genotype. In addition, 2 samples next to each other in the sample tray were manually exchanged by mistake, resulting in 4 discrepant genotypes. This was verified by repeat analysis of the stock DNA. Finally, 1 sample had a melting curve between wild-type and heterozygous curves and could not be genotyped. Subsequent targeted sequencing of its stock DNA revealed 80%–90% C allele and 10%–20% T allele (data not shown), suggesting a mixed DNA sample or product contamination. Overall, correct genotypes were obtained on the first pass 98.63% of the time, with 0.75% requiring 1 repeat and 0.63% of samples compromised by exchange or mixing. Number of clinical samples by locus and genotype that were correctly genotyped without repeats or additional analysis at Canon US Life Sciences. Table 2. Number of clinical samples by locus and genotype that were correctly genotyped without repeats or additional analysis at Canon US Life Sciences. Target . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 79/79 20/20 1/1 F5 c.1601G>A 75/75 22/22 3/3 MTHFR c.665C>T 54/56a,b 39/40b 4/4 MTHFR c.1286A>C 23/23 53/53 24/24 Target . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 79/79 20/20 1/1 F5 c.1601G>A 75/75 22/22 3/3 MTHFR c.665C>T 54/56a,b 39/40b 4/4 MTHFR c.1286A>C 23/23 53/53 24/24 a One sample had a melting curve between wild-type and heterozygous genotypes that precluded genotyping. Sequencing of the purified DNA stock of this sample showed about 90% C allele and 10% T allele (presumably resulting from DNA or amplicon contamination). The melting curves of the other 3 loci were successfully genotyped on the first run. b Two samples of MTHFR c.665C>T showed nonspecific amplification in their melting curves that precluded genotyping on the initial run. These samples were repeated once and correctly genotyped. Open in new tab Table 2. Number of clinical samples by locus and genotype that were correctly genotyped without repeats or additional analysis at Canon US Life Sciences. Target . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 79/79 20/20 1/1 F5 c.1601G>A 75/75 22/22 3/3 MTHFR c.665C>T 54/56a,b 39/40b 4/4 MTHFR c.1286A>C 23/23 53/53 24/24 Target . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 79/79 20/20 1/1 F5 c.1601G>A 75/75 22/22 3/3 MTHFR c.665C>T 54/56a,b 39/40b 4/4 MTHFR c.1286A>C 23/23 53/53 24/24 a One sample had a melting curve between wild-type and heterozygous genotypes that precluded genotyping. Sequencing of the purified DNA stock of this sample showed about 90% C allele and 10% T allele (presumably resulting from DNA or amplicon contamination). The melting curves of the other 3 loci were successfully genotyped on the first run. b Two samples of MTHFR c.665C>T showed nonspecific amplification in their melting curves that precluded genotyping on the initial run. These samples were repeated once and correctly genotyped. Open in new tab Number of clinical samples by locus and genotype that were correctly genotyped without repeats or additional analysis at the University of Utah. Table 3. Number of clinical samples by locus and genotype that were correctly genotyped without repeats or additional analysis at the University of Utah. . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 78/78 20/20 2/2 F5 c.1601G>A 72/75a,b 20/22a,c 3/3 MTHFR c.665C>T 55/56a 39/40a 4/4 MTHFR c.1286A>C 24/24 51/52c 24/24 . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 78/78 20/20 2/2 F5 c.1601G>A 72/75a,b 20/22a,c 3/3 MTHFR c.665C>T 55/56a 39/40a 4/4 MTHFR c.1286A>C 24/24 51/52c 24/24 a Genotypes at F5 c.1601G>A and MTHFR c.665C>T of 2 neighboring DNA samples appeared to be exchanged on analysis of the first run. After repeat analysis using the stock DNA samples, the initial exchange (which most likely occurred during manual loading of the 384-well plate) was confirmed. b Two samples did not amplify well at F5 c.1601G>A (low signal strength), precluding genotyping on the initial run. These samples were repeated once and correctly genotyped. c One F5 c.1601G>A and 1 MTHFR c.1286A>C could not be evaluated on the initial run because of irregular fluid flow in one cartridge channel. These samples were repeated once and correctly genotyped. Open in new tab Table 3. Number of clinical samples by locus and genotype that were correctly genotyped without repeats or additional analysis at the University of Utah. . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 78/78 20/20 2/2 F5 c.1601G>A 72/75a,b 20/22a,c 3/3 MTHFR c.665C>T 55/56a 39/40a 4/4 MTHFR c.1286A>C 24/24 51/52c 24/24 . Wild-type . Heterozygote mutant . Homozygote mutant . F2 c.*97G>A 78/78 20/20 2/2 F5 c.1601G>A 72/75a,b 20/22a,c 3/3 MTHFR c.665C>T 55/56a 39/40a 4/4 MTHFR c.1286A>C 24/24 51/52c 24/24 a Genotypes at F5 c.1601G>A and MTHFR c.665C>T of 2 neighboring DNA samples appeared to be exchanged on analysis of the first run. After repeat analysis using the stock DNA samples, the initial exchange (which most likely occurred during manual loading of the 384-well plate) was confirmed. b Two samples did not amplify well at F5 c.1601G>A (low signal strength), precluding genotyping on the initial run. These samples were repeated once and correctly genotyped. c One F5 c.1601G>A and 1 MTHFR c.1286A>C could not be evaluated on the initial run because of irregular fluid flow in one cartridge channel. These samples were repeated once and correctly genotyped. Open in new tab Discussion We used a prototype microfluidic instrument for rapid-cycle, serial PCR and HSM to genotype 100 samples at 2 laboratories (800 analyzed loci) with 100% instrument accuracy. The platform was designed to perform multiple sequential assays on 8 different samples (1 sample/channel). Within the 8-sample limit, the number of samples and positive and negative controls is flexible, although running >8 samples requires additional microfluidic cartridges and runs. For each serial assay, different PCR protocols can be run and the number of genotyping assays can be increased to at least 25 on existing instrumentation. Rapid-cycle PCR was introduced in 1990 (21–23) and defined as 30 cycles of PCR in 10–30 min. The microfluidic instrument presented here performs near the fast end of this range. Furthermore, with the microfluidics on this instrument, we have initial evidence that the time for PCR can be reduced even further. High-resolution melting (sometimes abbreviated HRM) was introduced in 2003 (4, 24). Accurate genotyping of the thrombophilia targets presented here has been reported on more conventional instruments (8, 25). The maximum speeds reported to date for high-resolution melting are 0.3 °C/s (26, 27). The prototype presented here routinely melts at 0.5 °C/s, 67% faster than previously reported rates. Because we have observed improvements in heterozygote detection at faster rates, we are introducing the new term, high-speed melting, and its abbreviation, HSM, to differentiate faster methods from slower instruments that typically perform high-resolution melting at around 0.01–0.04 °C/s (27). The prototype presented here combines and extends the speed advantages of both rapid-cycle PCR and high-resolution melting. The system is targeted toward analyzing a few DNA samples at multiple variant loci, not parallel analysis on many DNA samples. Use of internal temperature controls decreased the Tm SD from 0.18 to 0.07 °C, allowing wild-type and homozygous genotypes to be easily distinguished, similar to other studies (15). The Tm range of the 4 single nucleotide polymorphisms studied here was 76.6–79.6 °C, with the internal temperature control well outside this range at 85.0 °C to prevent interference. For amplicons with a Tm near to this internal control, a second internal control with lower Tm could be used. Many patients could benefit from more rapid genetic test results. One of the more compelling reasons for easy access and reduced turnaround time for genetic testing is posed by variant-specific drug treatments such as ivacaftor for some cystic fibrosis variants, including Gly551Asp (28). Early trials have shown improved lung function, suggesting that lung damage may be minimized if patients who bear the targeted variant can be identified at birth and treatment initiated. Another example is illustrated by the current practice of diagnosing inborn errors of metabolism such as medium-chain acyl coenzyme A dehydrogenase deficiency. This genetic disorder occurs in approximately 1 in 15 000 births in the US population (29) and is a low-volume test for laboratories equipped to perform it. Initial screening by mass spectrometry is confirmed with genotyping. Because treatment to avoid severe seizures, coma, or death consists of simply changing to a low-fat diet and avoiding periods of fasting, confirming newborns with this disease before hospital discharge can be lifesaving (30). Finally, rapid identification of variants that affect drug metabolism or confer drug resistance allows for better patient treatment. Identification of slow vs rapid metabolizers for cytochrome P-450 can be critical to achieve the correct dose of clopidogrel for treatment of percutaneous coronary syndromes (31) and may improve dosing of warfarin treatment (32). The lack of genetic testing at local laboratories also prevents identification of individuals affected with rare diseases during an opportune moment for counseling. Recently Evans et al. (33) argued that identification of individuals carrying rare mutations usually leads to familial evaluation, identifying carriers and additional affected individuals, thereby increasing the overall benefit. As new genetic information becomes available, the ability to rapidly develop and validate additional genetic tests on a fast serial platform is attractive. An additional benefit of serial PCR is automatic reflexing to additional assays depending on the results of a preceding test. This can provide “smart” throughput while eliminating generation of extraneous information. Such a simplified work flow does not require additional setup. For example, exon scanning with HSM can identify the presence of variants, followed by reflex genotyping for the most common variants in the region of the positive scan. Such an approach has been successfully demonstrated for PAH (phenylalanine hydroxylase) (34), CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] (1), and BRCA1 (breast cancer 1, early onset) (35). Also, reflex testing was used by Guedes et al. (36) by scanning multiple regions in signal transduction gene pathways associated with prevalent mutations in colorectal cancers to determine eligibility for anti–epidermal growth factor receptor therapy. Tumor mutation analysis can first look for the most prevalent mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog) codons 12/13 and then reflex to the next likely BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E (37). Several future enhancements can be envisioned to advance system function and performance. Genotyping can be automated in software rather than the visual calls used here. The Tm differences between wild-type and homozygous variant products are large enough (0.8–1.1 °C) and the precision good enough (SD 0.07 °C), that Tm values can be used for definitive genotyping. The pipette system can be augmented to make reagent setup completely automatic, reducing time and operator error. Reduced time for reagent mixing should also limit nonspecific amplification, and the amount of DNA can be decreased. Improving thermal insulation around the microfluidic chip will improve performance by limiting temperature gradients across channels, especially the outer channels. Newly designed chips with additional heaters on the outside of channels 1 and 8 can reduce the thermal gradient observed, and fewer no-template controls can be used. Better temperature precision will improve the resolution between wild-type and homozygous samples, hopefully allowing the discrimination of class 3 and 4 variants (8) and difficult small insertions/deletions. Alternatively, unlabeled probes (9) or snapback primers (10) can be used to genotype difficult variants and can be performed on the same platform. In conclusion, serial PCR and HSM improve work flow for low-volume genetic tests. When combined with rapid PCR and HSM, turnaround time is reduced and multiple tests can be performed on the same samples. The platform also has the potential for customized repeat and reflexive testing to minimize sample reprocessing and generation of extraneous data. 5 Nonstandard abbreviations: HSM high-speed melting ARUP Associated Regional and University Pathologists Tm melting temperature LED light-emitting diode HRM high-resolution melting. 6 Human genes: F5 coagulation factor V (proaccelerin, labile factor) F2 coagulation factor II (thrombin) MTHFR methylenetetrahydrofolate reductase (NAD(P)H) PAH phenylalanine hydroxylase CFTR cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) BRCA1 breast cancer 1, early onset KRAS Kirsten rat sarcoma viral oncogene homolog BRAF v-raf murine sarcoma viral oncogene homolog B. " Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article. " Authors' Disclosures or Potential Conflicts of Interest:Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: " Employment or Leadership: S. Sundberg, Canon US Life Sciences; C.T. Wittwer, BioFire Diagnostics and Clinical Chemistry, AACC; R. Howell, Canon US Life Sciences; J. Huuskonen, Canon US Life Sciences; H. Stiles, Canon US Life Sciences; I.T. Knight, Canon US Life Sciences. " Consultant or Advisory Role: None declared. " Stock Ownership: None declared. " Honoraria: None declared. " Research Funding: Grant from Canon US Life Sciences to the University of Utah, C. Wittwer, PI. " Expert Testimony: None declared. " Patents: S. Sundberg, application no. PCT/US2011/050104; C.T. Wittwer, patent no. 7803551; R. Palais, patent nos. 8068992 B2 and 8296074 B2; I.T. Knight, patent nos. US7915030B2, US20110262316A1, and US20120288865A1. " Other Remuneration: S. Sundberg, travel between University of Utah and Rockville, MD; H. Stiles, Canon US Life Sciences. " Role of Sponsor: The funding organizations played a direct role in the review and interpretation of data. Acknowledgments The authors thank ARUP laboratories for providing the deidentified samples and Ling Xu, Rob Troyan, and Sandhya Patel from Canon US Life Sciences for providing technical assistance. References 1. Zhou L , Palais RA, Ye F, Chen J, Montgomery JL, Wittwer CT. Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene . 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TI - Microfluidic Genotyping by Rapid Serial PCR and High-Speed Melting Analysis
JF - Clinical Chemistry
DO - 10.1373/clinchem.2014.223768
DA - 2014-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/microfluidic-genotyping-by-rapid-serial-pcr-and-high-speed-melting-MwPCLF70Hn
SP - 1306
VL - 60
IS - 10
DP - DeepDyve
ER -