Improved Performance of the Maxwell® 16 Low Elution Volume System for Forensic Casework

Introduction

Previously, we described a medium-throughput, automated method to purify DNA from casework samples using the DNA IQ™ System and Maxwell® 16 Instrument and showed that the performance of this method compared favorably to that of commercially available DNA purification instruments ⁽¹⁾ . We also introduced several disposable, plastic components of the Maxwell® 16 System, including the original reagent cartridge and plunger for the DNA IQ™ Casework Sample Kit for Maxwell® 16. However, subsequent testing at Promega demonstrated that this DNA isolation method performed inconsistently between lots, frequently with lower DNA isolation efficiency than that of the DNA IQ™ System chemistry performed manually. Therefore, we performed experiments to determine if changes to the DNA IQ™ chemistry and Maxwell® 16 DNA isolation robotic method would improve DNA yield. Knowing that plastics can affect biomolecular interactions ⁽²⁾ , we also investigated the role of plasticware provided with the DNA IQ™ Casework Sample Kit for Maxwell® 16 in the performance variation.

We identified a material change in a single component that improved DNA isolation efficiency so that it was equal to or greater than that of the manual DNA IQ™ protocol and consistent from lot to lot of the underlying material. This increase was achieved without changing the Maxwell® 16 Instrument firmware or DNA IQ™ chemistry. In this article, we describe the results of changing the plastic used to construct the LEV Plungers and the improved DNA yields from different sample types. These improved LEV Plungers were incorporated into the new DNA IQ™ Casework Pro Kit for Maxwell® 16 (Cat.# AS1240), which also features improved cartridge design and streamlined sample-processing protocols. We show that the resulting purification chemistry is consistent and exhibits a DNA isolation efficiency with certain sample types comparable to that of organic extraction, which is widely recognized as a gold standard in forensic sample preparation.

Note: Yield is a composite of two separate processes: Extraction of sample from the solid support and isolation of nucleic acids using the DNA IQ™ chemistry. The changes described here affect isolation efficiency, which should be independent of sample type.

"We developed an improved LEV Plunger that increases DNA isolation efficiency of the DNA IQ™ Casework Sample Kit for Maxwell® 16 with no changes to the established DNA IQ™ chemistry or Maxwell® 16 Instrument firmware."

Improved Design of the LEV Plunger

During experiments to determine the root cause of the performance variation, we identified a manufacturing material for the LEV plunger that increased isolation efficiency and reduced variability between runs. Because LEV Plungers made from this new material are almost identical to the original LEV Plungers in dimensions (Figure 1), the improved LEV Plungers can be substituted into the DNA IQ™ Casework Sample Kit for Maxwell® 16 without any changes to the extraction method, DNA IQ™ chemistry or Maxwell® 16 Instrument firmware to achieve greater DNA yield from samples.

Figure 1. The original LEV Plunger (left) and new LEV Plunger.

Improved DNA Isolation Efficiency on the Maxwell® 16 Instrument with the New LEV Plungers

To illustrate the advantage of this new material, we isolated DNA from identical liquid blood samples, pooled touch samples and blood swabs using the Maxwell® 16 Instrument and LEV Plungers constructed from either the old or new material. For comparison, we also isolated DNA using the manual DNA IQ™ System protocol. Prior to DNA purification, samples were preprocessed as follows:

Liquid blood samples: For each replicate sample, 0.5µl of whole blood was combined with 10µl of Proteinase K Solution (18mg/ml) and 89.5µl of Incubation Buffer from the Tissue and Hair Extraction Kit (for use with DNA IQ™) (Cat.# DC6740). Samples were incubated for 1 hour at 56°C, then combined with 300µl of DNA IQ™ Lysis Buffer and vortexed thoroughly.

Pooled touch samples: Each of 18 forensic touch samples (swabs collected from keyboards, telephone handles and personal items in the laboratory) was combined with 10µl of Proteinase K Solution (18mg/ml) and 90µl of Incubation Buffer from the Tissue and Hair Extraction Kit (for use with DNA IQ™). Samples were incubated for 1 hour at 56°C, then combined with 300μl of DNA IQ™ Lysis Buffer and vortexed thoroughly. Samples were centrifuged through a DNA IQ™ Spin Basket (Cat.# V1221) at 14,000 × g, and the resulting lysates were combined into a single pooled lysate. Equal volumes of this pooled lysate were processed.

Blood on swabs: 50µl of a 1:50 dilution of whole blood in phosphate-buffered saline was allowed to dry overnight on a swab. Each swab was combined with 10µl of Proteinase K Solution (18mg/ml) and 90µl of Incubation Buffer from the Tissue and Hair Extraction Kit (for use with DNA IQ™). Samples were incubated for 1 hour at 56°C, then combined with 300μl of DNA IQ™ Lysis Buffer and vortexed thoroughly. Samples then were centrifuged through a DNA IQ™ Spin Basket at 14,000 × g.

These lysates were processed manually using the DNA IQ™ System (Cat.# DC6700), as directed in the DNA IQ™ System—Small Sample Casework Protocol #TB296, or processed using the DNA IQ™ Casework Sample Kit for Maxwell® 16. Half of the samples processed using the Maxwell® 16 Instrument were processed with LEV Plungers manufactured using the new material, while the other half were processed with the original LEV Plungers. All DNA samples were eluted in 50µl volumes. The resulting DNA eluates were quantified with the Plexor® HY System (Cat.# DC1001) to estimate DNA yield. Results are shown in Figure 2.

Figure 2. Improved DNA isolation efficiency on the Maxwell® 16 Instrument with the improved LEV Plungers.

DNA was isolated using the DNA IQ™ System, performed manually using the DNA IQ™ System—Small Sample Casework Protocol, or the DNA IQ™ Casework Sample Kit for Maxwell® 16 with the original and improved LEV Plungers as described in the text. Panel A. DNA yield from 0.5 µl of whole blood. Panel B. DNA yield from 1 µl of blood on a swab. Panel C. DNA yield from pooled touch samples. Error bars represent one standard error of the mean (SEM).

Comparison of DNA IQ™ Casework Pro Kit for Maxwell® 16 and Organic Extraction

We compared DNA yields obtained from liquid blood using the Maxwell® 16 Instrument, new LEV Plungers and Maxwell® 16 LEV Cartridges to yields obtained using organic extraction. Liquid blood was diluted 1:4 in phosphate-buffered saline, then serially diluted fivefold. Twenty microliters of each dilution was processed in triplicate using the DNA IQ™ Casework Pro Kit for Maxwell® 16 (i.e., the improved LEV Plungers) or organic extraction as indicated below.

DNA IQ™ Casework Pro Kit for Maxwell® 16: We combined 20µl of diluted blood with 10µl of Proteinase K Solution (18mg/ml) and 270µl of Incubation Buffer from the Tissue and Hair Extraction Kit (for use with DNA IQ™), then incubated samples for 1 hour at 70°C as per the DNA IQ™ Casework Pro Kit for Maxwell® 16 protocol #TM332. The resulting extract was combined with 150µl of DNA IQ™ Lysis Buffer, vortexed, placed directly into the DNA IQ™ Casework Pro Kit for Maxwell® 16 LEV Cartridge and processed using version 4.71 of the forensic method for the Maxwell® 16 Instrument LEV. Volumes of eluted samples were measured, and DNA was quantified using the Plexor® HY System.

Organic Extraction: We combined 20µl of diluted blood with 290µl of an organic stain extraction buffer [10mM EDTA, 10mM Tris, 100mM NaCl, 2% SDS] and 10µl of Proteinase K Solution (18mg/ml), and incubated samples for 4 hours at 56°C. We then added a phenol:chloroform:isoamyl alcohol solution [25:24:1, saturated with 10mM Tris (pH 8.0) and 1mM EDTA (Sigma Cat.# P2069)], followed by concentration of samples with a commercially available concentrator unit (Ultracel YM-100, Millipore Cat.# 42424). Volumes of eluted samples were measured, and DNA was quantified using the Plexor® HY System.

Results of DNA isolations using organic extraction and the DNA IQ™ Casework Pro Kit for Maxwell® 16 were comparable and showed a linear relationship between DNA yield and blood volume input (Figure 3).

Figure 3. Comparative DNA isolation from whole blood using the DNA IQ™ Casework Pro Kit for Maxwell® 16 and organic extraction.

Serial dilutions of whole blood were processed in triplicate using the DNA IQ™ Casework Pro Kit for Maxwell® 16 or a traditional organic extraction protocol. Average DNA yield was measured using the Plexor® HY System and is plotted across the titration series.

Comparison of the DNA IQ™ Casework Pro Kit for Maxwell® 16 and EZ1™ DNA Investigator Kit

Forensic casework presents a variety of sample types, including biological stains such as blood or other biological fluids dried on a physical matrix. Samples also may consist of touch evidence swabs, which are collected using moistened swabs to collect cellular material from a surface. We compared DNA yields from mock casework swab samples using the DNA IQ™ Casework Pro Kit for Maxwell® 16 and Qiagen EZ1™ DNA Investigator Kit.

To accurately gauge the relative efficiencies of different extraction methods, it was necessary to deposit an equal amount of sample onto swabs prior to DNA extraction. We diluted liquid blood 1:50 in phosphate-buffered saline and spotted 50µl onto each cotton swab. Swabs were dried overnight at room temperature. DNA was extracted using the DNA IQ™ Casework Sample Kit for Maxwell® 16 or Qiagen EZ1™ DNA investigator kit as described below. DNA was quantified with the Plexor® HY System, and DNA concentrations were multiplied by recovered elution volumes to determine DNA yield for each sample type.

DNA IQ™ Casework Pro Kit for Maxwell® 16: Each swab was incubated with 90µl of Incubation Buffer from the Tissue and Hair Extraction System (for use with DNA IQ™) and 10µl of 18mg/ml Proteinase K for 1 hour at 70°C. Each sample was combined with 300µl of DNA IQ™ Lysis Buffer, then centrifuged through a DNA IQ™ Spin Basket at 14,000 × g for 2 minutes in a microcentrifuge to separate the lysate and solid support. All lysates were placed into the Maxwell® 16 LEV cartridge, loaded with the improved LEV plunger and processed using version 4.56 of the LEV Forensic Method. DNA was eluted in 50µl of Elution Buffer.

EZ1™ DNA Investigator Kit: Each blood swab was incubated as described in the EZ1™ DNA Investigator Handbook (including pretreatment for forensic surface and contact swabs with an additional 5-minute incubation step at 95°C, as recommended for maximum yield), followed by the trace protocol using the EZ1™ DNA investigator card installed on a BioRobot® EZ1™ workstation. DNA was eluted in 50µl of TE buffer as per the manufacturer’s recommendation.

DNA yield using the improved Maxwell® 16 LEV Plungers was higher than that obtained using the Qiagen EZ1™ DNA Investigator's Kit (Figure 4).

Figure 4. Comparison of the DNA IQ™ Casework Pro Kit for Maxwell® 16 with the new LEV Plungers and Qiagen EZ1™ DNA Investigator’s Kit.

Whole blood was diluted 1:50 in phosphate-buffered saline, and 50µl was placed on a swab and dried. DNA was isolated per the manufacturer's recommendations. DNA was quantified using the Plexor® HY System. Error bars represent one standard error of the mean (SEM).

Conclusions

We developed an improved LEV Plunger that results in increased DNA isolation efficiency on the Maxwell® 16 Instrument. This change is limited to the plunger and did not require any change to the established DNA IQ™ chemistry or Maxwell® 16 Instrument firmware. Use of the new LEV Plungers yielded DNA isolation efficiency comparable to that of organic extraction when performed by an experienced operator. These improved LEV Plungers were incorporated into the new DNA IQ™ Casework Pro Kit for Maxwell® 16, which is now commercially available.

Acknowledgments

The authors acknowledge the able assistance of Megan Hornung with research support, Erin McCombs and Elaine Schenborn in the development of the DNA IQ™ Casework Pro Kit for Maxwell® 16 kit, and Michelle Mandrekar for a critical reading of the manuscript.

Related Products

• Maxwell® FSC Instrument
• Maxwell® FSC DNA IQ™ Casework Kit
• DNA IQ™ Kits for Maxwell® 16

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• The Maxwell® 16 Low Elution Volume System for Forensic Casework: Implementation and Routine Use in a Forensic Laboratory
• The Maxwell® 16 Low Elution Volume System for Forensic Casework
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Article References

1. Mandrekar, P. et al. (2007) The Maxwell® 16 low elution volume system for forensic casework. Profiles in DNA 10(2),
2. McDonald, G.R. et al. (2008) Bioactive contaminants leach from disposable laboratory plasticware. Science 322, 917.

How to Cite This Article

Scientific Style and Format, 7th edition, 2006

Mandrekar, P.V. et al. Improved Performance of the Maxwell® 16 Low Elution Volume System for Forensic Casework. [Internet] 2010. [cited: year, month, date]. Available from: https://www.promega.com/es-es/resources/profiles-in-dna/improved-performance-of-the-maxwell-16-low-elution-volume-system-for-forensic-casework/

American Medical Association, Manual of Style, 10th edition, 2007

Mandrekar, P.V. et al. Improved Performance of the Maxwell® 16 Low Elution Volume System for Forensic Casework. Promega Corporation Web site. https://www.promega.com/es-es/resources/profiles-in-dna/improved-performance-of-the-maxwell-16-low-elution-volume-system-for-forensic-casework/ Updated 2010. Accessed Month Day, Year.