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Letters |
1 ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108
2 ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108
3 Department of Pathology, University of Utah School of Medicine, 50 North Medical Dr., Salt Lake City, UT 84132
aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108. Fax 801-584-5207; e-mail lyone{at}aruplab.com.
To the Editor:
We evaluated the MagNA Pure LC System (Roche Applied Science) for automated DNA extraction for use in mutation detection and allele quantification. The system uses magnetic silica beads that bind DNA to a silica surface and transfer DNA through various steps of the extraction process. This system can be linked to LightCycler assays for further automation. The LightCycler is an enclosed thermocycler with continuous monitoring for PCR quantification and allele discrimination (1)(2)(3). Combined MagNA Pure/LightCycler automation of sample preparation, amplification, and detection has the potential to reduce human error in sample tracking and to standardize DNA extraction and inoculation into the PCR mixture. The MagNA Pure/LightCycler system has been evaluated for infectious disease applications, detecting PCR product by crossing point (4)(5). In this study we compared the MagNA Pure LC DNA extraction with the Qiagen 9604 BioRobot (Qiagen) for real-time mutation detection and allele quantification.
We evaluated 280 samples with the MagNA Pure System and Qiagen 9604 BioRobot under an Internal Review Board-approved protocol for instrument validation. Genomic DNA was extracted from 200 µL of EDTA-anticoagulated whole blood by use of the MagNA Pure LC DNA Isolation Kit I (Roche Diagnostics), with its high-performance DNA protocol, and by the QIAamp 9604 DNA Blood BioRobot Kit (Qiagen) according to manufacturer’s instructions. Both methods initially use red blood cell lysis and a proteinase K procedure, and the DNA binds to a silica-based support. The MagNA Pure separates contaminants from DNA by use of magnetic beads, whereas the 9604 BioRobot uses a vacuum manifold.
Genotypes were evaluated for both extraction systems by LightCycler-based assays developed for factor V Leiden (2), factor II (manufacturer’s directions), hemochromatosis (6), and the methylenetetrahydrofolate reductase gene, MTHFR (3). Control DNA (wild-type, heterozygous, and homozygous) was obtained from previously tested samples extracted by the Qiagen 9604 BioRobot or by phenol–chloroform extraction (7). DNA samples genotyped for factor V Leiden (n = 106) were further evaluated. DNA yield and purity (A260/A280) were assessed by spectrophotometry (Pharmacia Biotech Inc.). LightCycler-specific parameters, including crossing points, melting temperatures (Tm), and areas of derivative melting curves, were determined. Sample types were also tested. Finally, technical time was compared for the two methods.
The performance characteristics of the two methods are summarized in Table 1
. Comparison of 106 identical blood samples from each extraction method showed that the MagNA Pure and the 9604 BioRobot were similar in DNA yields, although the DNA purity was higher in samples extracted by the MagNA Pure. Genotypes for all assays agreed completely. The few extraction failures in each system were different samples, indicating that failure rate was not sample-specific.
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LightCycler-specific results were determined by comparing identical samples extracted by Qiagen and MagNA Pure tested side by side within a LightCycler run to eliminate run-to-run variability. The crossing point represents the point at which amplification of DNA is detected above background fluorescence and is used for real-time quantification. Variations in crossing point reflect different DNA quantities between samples.
A strength in mutation detection by melting cure analysis is the ability to differentiate between mutations within the region of the probe based on probe Tm (6)(8)(9). This allows analysis of multiple mutations and reduces false positives attributable to unknown base alterations near the mutation of interest. A Tm difference as little as 0.8 °C resulting from a novel mutation has been described (8). Sample-to-sample concentration differences as well as differences in elution buffers may cause shifts in Tm, but the difference between the Tm of the wild-type allele and that of the mutant allele (
Tm) should remain constant between samples. For this reason, Tms may indicate alternative mutations better than Tm alone (10).
Five heterozygous samples were detected in two separate runs. Although the sample size was limited, statistical analysis using an F-test indicated that the Tms for the MagNA Pure system were more consistent than those for the Qiagen BioRobot system (P <0.001). The difference in CVs between the methods may be attributable to differences in sample purity.
End-point quantification has also been reported for the LightCycler. Examples of these are competitive PCR assays (11)(12) and assays using reference genes (13). A ratio between areas of derivative melting curves can be used to detect duplications or deletions. Consistencies in the relative area of each allele are necessary for this type of quantification. Areas for wild-type and mutant alleles were determined, and the ratios of allele areas (Area 1/Area 2) for heterozygous samples were calculated. The optimal allele area ratio for heterozygous samples is 1. Statistical analysis using an F-test indicated that the Roche MagNA Pure system had less variation than the Qiagen 9604 BioRobot system (P = 0.001).
We also tested sodium citrate and heparin collection tubes. All samples could be amplified, although heparin samples required the "fast" protocol as opposed to the "high performance" protocol on the MagNA Pure. The fast protocol uses a shorter lysis incubation time (14 vs 7 min) and DNA binding time (12 vs 3 min) than the high-performance protocol. Both MagNA Pure protocol options are included with the instrument. For heparin samples, fluorescence (as determined by allele area) was much lower from Qiagen-extracted samples than from phenol–chloroform- or MagNA Pure-extracted samples (P <0.05, two-tailed t-test). Sodium citrate samples extracted by both the MagNA Pure and the Qiagen methods had less fluorescence than those extracted with phenol–chloroform (P <0.001).
Finally, hands-on technical time was evaluated for the MagNA Pure and Qiagen samples. Technical time for the MagNA Pure was 
20 min for 32 samples. The Qiagen 9604 BioRobot required 1 h for 96 samples. Thus, technical time was similar with both systems.
For clinical use, extraction methods should produce DNA with concentrations >50 mg/L and purities showing an A260/A280 ratio >1.8. A lower yield could increase the sample volume needed, the reextraction rate, or the frequency of resampling. For the LightCycler, we accept a Tm ± 0.25 °C of the within-run average for clinical samples. A high CV for Tms may increase the number of samples falling outside the established parameters and lead to a need for reextraction. Additionally, novel mutations may be missed. High CVs for allele areas would decrease the accuracy of allele quantification assays. When evaluated for clinical use, the MagNA Pure DNA met the requirements for sample yield and purity and exceeded the standards for Tm and allele area ratios.
Acknowledgments
This project was supported in part by Roche Molecular Biochemicals (instrument loan and reagents) and ARUP Institute for Clinical and Experimental Pathology.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Millson, G. Pont-Kingdon, S. Page, and E. Lyon Direct Molecular Haplotyping of the IVS-8 Poly(TG) and PolyT Repeat Tracts in the Cystic Fibrosis Gene by Melting Curve Analysis of Hybridization Probes Clin. Chem., September 1, 2005; 51(9): 1619 - 1623. [Abstract] [Full Text] [PDF]
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