Automated Extraction of High Molecular Weight DNA

Requirements placed on samples to be processed continue to grow. In both academic settings and industry, automation solutions are becoming more and more important.

The purification of genomic DNA (gDNA) has provided the foundation for a multitude of subsequent processes where the purity and quantity of isolated DNA is vital.

Traditional extraction methods make use of costly reagents and rely on the effective handling of small sample quantities. The resulting purification procedures are repeated several times, with each iteration increasing the risk of sample material loss.

Analytik Jena‘s SmartExtraction is an innovative technology designed for the extraction of high molecular weight DNA. This technology is the cornerstone of a fundamentally new approach to the automated isolation and purification of nucleic acids.

SmartExtraction utilizes a combination of patented extraction chemistry (DC Technology®) with an intelligent ‘Smart Modified Surface’. This approach makes the entire automated workflow of DNA extraction simpler, providing previously unattainable levels of yield, efficiency, and DNA quality.

SmartExtraction makes it possible to obtain very large quantities of DNA, and it is feasible to isolate high molecular weight DNA (200 kb - >500 kb) via an automated process. The technology’s ‘Smart Modified Surface’ also allows users to map the whole extraction process inside a pipette tip, using basic pipetting steps.

SmartExtraction does not require the use of ion exchangers, phenol/chloroform, filter columns, or filter plates. Furthermore, the suspension of magnetic or paramagnetic particles is not required to bind the DNA. Pre-filled and sealed reagent plastics simplify the handling process and these can, in analogy to the functionalized pipette tips, be used directly within the CyBio® FeliX pipetting system.

When applying SmartExtraction for the automated extraction of nucleic acids, the CyBio® FeliX can be employed with 96 samples in parallel. Protocols for different starting materials are available, including smart Blood DNA Midi prep. This technology is especially useful in meeting increasing demands for automated liquid handling.

The CyBio® FeliX is a fully automated, flexible pipetting system able to accommodate between 1 and 384 channels in the volume range of 1 to 1000 µl. As well as accurate, parallel transfer in the 96- and 384-well format, pipetting can also be performed by single-channel, row or column. Within each pipetting routine, the most appropriate accessory will be automatically determined and swapped in.

CyBio® FeliX offers the utmost flexibility with minimal space requirements, largely due to its distinctive design with 12 positions on two levels. Its modular approach allows application-specific configurations with pre-configured and optimized pipetting routines to be added at any time.

CyBio® FeliX Basic Unit

Figure 1. CyBio® FeliX Basic Unit.

Material

  • CyBio® FeliX Basic Unit (OL5015-24-100, Analytik Jena AG)
  • CyBio® FeliX head R 96/1000 µl (OL3316-14-950, Analytik Jena AG)
  • 96-channel magazine; head R 96/1000 µl (OL3810-13-024, Analytik Jena AG)
  • smart Blood DNA Midi prep (a96) (845-ASP-1296096, Analytik Jena AG)
  • thermal mixer (e.g. BioShake iQ, 848-1808-0506, Analytik Jena AG)
  • centrifuge (e.g. Eppendorf 5424R)
  • gel electrophoresis system (e.g. Compact M electrophoresis chamber for agarose gels, 846-025-200, Analytik Jena AG)
  • 1 x PBS buffer (137 mM NaCl; 2,7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄)
  • ddH₂O (S15-012, GE Healthcare)
  • LE agarose (Biozym, 840004)
  • 1 x TBE buffer, pH 8.0
  • spectrophotometer (ScanDrop® 250, Analytik Jena AG)
  • Roti®-Load DNA with glycerol (Roth, X904.1)
  • whole blood (stabilized with EDTA, stored for 2 months at -80 °C)

Methods

Lysis of Erythrocytes

A total of 3 ml of whole blood was used from each donor. Both the lysis of erythrocytes and pelleting of nucleated blood cells was undertaken in line with the guidance in the manual (Kit: smart Blood DNA Midi prep(a96)). Following the pelleting process, nucleated blood cells were resuspended in 120 µl 1 x PBS.

Enzymatic Lysis (Proteolysis) and Extraction

Proteolytic lysis of the cells was done externally, with 200 µl of a lysis buffer (Lysis Solution CBV) and 30 µl Proteinase K added to the resuspended cells before these were incubated for 30 minutes using the BioShake iQ at 800 rpm and 55 °C. Lysate was then transferred into the reagent plate before being placed onto device position 10 of the CyBio® FeliX.

The automatic processing and elution of DNA samples was undertaken in line with guidance from the smart Blood DNA Midi prep(a96) protocol. The kit’s reagent plates necessary for automated DNA extraction were pre-filled with all the reagents.

Verification of DNA Extraction

Extracted DNA was successfully verified using agarose gel electrophoresis. This process involved the application of 10 µl of each eluate and 7 µl of the DNA ladder to a 0.8% TBE gel which contained 5 µl of ethidium bromide for every 100 ml of agarose gel.

Electrophoretic separation occurred in a horizontal gel electrophoresis system (Analytik Jena AG). This was achieved through the constant application of 127 mA for optimal running conditions.

UV light was used to visualize the resulting DNA bands, and both the yield and quality of the DNA were ascertained using a spectrophotometer (ScanDrop® 250, Analytik Jena AG), with 4 µl of each eluate filled into a suitable CHIPCUVETTE® and measured by a path length of 1.0 mm.

CyBio® FeliX deck layout for automated extraction using smart Blood DNA Midi prep (a96).

Figure 2. CyBio® FeliX deck layout for automated extraction using smart Blood DNA Midi prep (a96).

Results and discussion

A combination of smart Blood DNA Midi prep (a96) and a CyBio® FeliX was employed to automatically extract genomic DNA (gDNA) from 10 different whole blood samples (3 ml).

Agarose gel electrophoresis (Figure 3) and spectrophotometric measurements (Table 1) were used in determining the yield and quality of this isolated DNA. Table 1 displays both the purity (A₂₆₀:A₂₈₀ and A₂₆₀:A₂₃₀) and concentration of the extracted DNA.

Concentration and purity measurements were completed using Analytik Jena AG’s ScanDrop® 250 instrument. The A₂₆₀:A₂₈₀ and A₂₆₀:A₂₃₀ evaluation indicated that the extracted DNA was of excellent quality.

This extraction method enabled a high yield of high molecular weight DNA to be isolated. Samples were distributed randomly on the plate to demonstrate the reproducibility and comparability of sample preparation within the reaction plate.

Results of horizontal agarose gel electrophoresis, gDNA from whole blood samples (pelleted nucleated cells), extracted by means of an automated process using smart Blood DNA Midi prep (a96) in combination with the CyBio® FeliX pipetting system. Visualized is a used quantity of 10 μl of eluate on a 0.8 % agarose gel with added ethidium bromide. Column 1: DNA ladder; Columns 2 – 10: DNA extracted from nucleus containing cells from 3 ml whole blood; Column 11 : negative control (CO)

Figure 3. Results of horizontal agarose gel electrophoresis, gDNA from whole blood samples (pelleted nucleated cells), extracted by means of an automated process using smart Blood DNA Midi prep (a96) in combination with the CyBio® FeliX pipetting system. Visualized is a used quantity of 10 μl of eluate on a 0.8 % agarose gel with added ethidium bromide. Column 1: DNA ladder; Columns 2 – 10: DNA extracted from nucleus containing cells from 3 ml whole blood; Column 11: negative control (CO).

Table 1. Results of the gDNA extraction from whole blood samples, spectrophotometrically determined using a ScanDrop® 250.

Lane Whole blood [ml] A260:A280 A260:A230 Conc. [ng/µl] Yield [µg]
1 DNA ladder
2 3 ml 1.8 2.4 188 94
3 3 ml 1.8 2.3 175 87.5
4 3 ml 1.8 2.6 216 108
5 3 ml 1.8 2.3 229 114.5
6 3 ml 1.8 2.2 213 106.5
7 3 ml 1.8 2.7 194 97
8 3 ml 1.8 2.3 216 108
9 3 ml 1.8 2.8 178 89
10 CO 0.0 0.0

 

Summary

SmartExtraction offers a novel and innovative means of  isolating nucleic acids. This technology can be efficiently and elegantly automated using the flexible CyBio® FeliX pipetting system, enabling isolation of up to 96 samples in parallel. It is possible to achieve high quality, reproducible pipetting results and the isolation of a high yield of high molecular weight DNA.

The modular system means that the degree of automation can be easily configured by the customer, while the flexible automation solution helps improve reproducibility while increasing the efficiency of lab processes. The CyBio® FeliX can also be utilized for a range of other downstream routine liquid handling tasks.

Overall, the automatic extraction of DNA for downstream processes results in straightforward, parallel sample preparation with maximum consistency and minimum effort.

About Analytik Jena US

Analytik Jena is a provider of instruments and products in the areas of analytical measuring technology and life science. Its portfolio includes the most modern analytical technology and complete systems for bioanalytical applications in the life science area.

Comprehensive laboratory software management and information systems (LIMS), service offerings, as well as device-specific consumables and disposables, such as reagents or plastic articles, complete the Group’s extensive range of products.

About life science

The Life Science product area demonstrates the biotechnological competence of Analytik Jena AG. We provide a wide product spectrum for automated total, as well as individual solutions for molecular diagnostics. Our products are focused to offer you a quality and the reproducibility of your laboratory results.

This will surely ease your daily work and speed up your work processes in a certain way. All together we support you through the complete process of the lab work. Besides we offer customized solutions and are able to adapt our products to your needs. Automated high-throughput screening systems for the pharmaceutical sector are also part of this segment’s extensive portfolio.

About analytical instrumentation

Analytik Jena has a long tradition in developing high-performance precision analytical systems which dates back to the inventions made by Ernst Abbe and Carl Zeiss. We have grown to become one of the most innovative manufacturers of analytical measuring technology worldwide.

Our business unit Analytical Instrumentation offers excellent competencies in the fields of optical spectroscopy, sum parameters and elemental analysis. Being proud of our core competency we grant all our customers a long-term warranty of 10 years for our high-performance optics.

About lab automation

With more than 25 years of market experience, Analytik Jena with its CyBio® Product Line is a leading provider for high quality liquid handling and automation technologies. In the pharmaceutical and life science industries, our products enjoy the highest reputation for precision, reliability, robustness and simplicity.

Moreover, the Automation Team designs, produces and installs fully automated systems tailored to our clients' application, throughput and capacity requirements. From stand-alone CyBio® Well up to fully customized robotic systems we handle your compounds, biomolecules and cells with great care.


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Last updated: Sep 29, 2020 at 3:28 AM

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