Extracting High Molecular Weight DNA Through Automation

Automated solutions are taking on ever growing importance. There are requirements placed on samples to be processed that are steadily increasing, both in academic settings as well as in the industry. Purification of genomic DNA (gDNA) is where it all begins for a large amount of consequent processes where the number and purity of the isolated DNA is vital.

Conventional extraction methods utilize expensive reagents and are based on the handling of small sample quantities as well as on the resulting purification procedures, which are repeated multiple times. Additionally, this increases the risk of losing valuable sample material.

SmartExtraction is Analytik Jena‘ s state-of-the-art technology for the extraction of high molecular weight DNA. This technology forms the basis of a fundamentally new method for the automated isolation and purification of nucleic acids.

SmartExtraction combines the patented extraction chemistry (DC Technology®) with a smart surface, called “Smart Modified Surface.” SmartExtraction significantly simplifies the entire automated workflow of DNA extraction and setting new standards in terms of efficiency, yield and quality of the DNA.

It is possible to gather particularly large quantities of DNA. In addition, it is feasible to isolate high molecular weight DNA (200 kb - >500 kb) using an automated process. The “Smart Modified Surface” also facilitates mapping of the entire extraction process inside a pipette tip via simple pipetting steps.

The SmartExtraction technology does not need phenol/chloroform, ion exchangers, filter columns or filter plates. In addition, a suspension of magnetic or paramagnetic particles is not needed to bind the DNA. It is made easier to handle through pre-filled and sealed reagent plastics, and can, in likeness to the functionalized pipette tips, be used straight onto the CyBio® FeliX pipetting system.

For automated extraction of nucleic acids applying smartExtraction the CyBio® FeliX can be used for 96 samples in parallel. Next to smart Blood DNA Midi prep additional protocols for different starting materials are available. The technology is especially suited to meet the growing demand for the automated handling of liquids.

A flexible, completely automated pipetting system, CyBio® FeliX has 1 to 384 channels in the volume range from 1 to 1000 µl. Along with an extremely precise, parallel transfer in the 96 and 384 well format, pipetting is possible through singlechannel, column or row.

The suitable accessory is mechanically chosen and altered within a pipetting routine. This product has a unique design with 12 positions on 2 levels and combines maximum flexibility with the least space requirements. Application specific configurations can be added whenever needed due to the modular concept of CyBio® FeliX. The emphasis continuously lies on the application with foundations of pre-configured and optimized pipetting routines.

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
  • Centrifuge, e.g. Eppendorf 5424R
  • Thermal mixer, e.g. BioShake iQ, 848-1808-0506, Analytik Jena AG
  • 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
  • 1 x TBE buffer, pH 8.0
  • LE agarose: Biozym, 840004
  • Roti®-Load DNA with glycerol: Roth, X904.1
  • Spectrophotometer: ScanDrop® 250, Analytik Jena AG
  • hole blood: stabilized with EDTA, stored for 2 months at -80 °C

Methods

Lysis of Erythrocytes

Each donor gave whole blood, and 3ml of this was used from each donor. Lysis of erythrocytes and pelleting of nucleated blood cells was completed in agreement with the manual information (Kit: smart Blood DNA Midi prep(a96)). Subsequently, the nucleated blood cells were totally resuspended in 120 µl 1 x PBS.

Enzymatic Lysis (proteolysis) and Extraction

Proteolytic lysis of the cells was completed externally, and 200 µl of a lysis buffer (Lysis Solution CBV) and 30 µl Proteinase K were added to the resuspended cells. Incubation for 30 min on the BioShake iQ for 800 rpm at 55 °C followed this, and the lysate was relocated into the reagent plate and positioned onto device position 10 of the CyBio® FeliX.

Automatic processing and elution of the DNA samples were done in accordance to the information noted in the smart Blood DNA Midi prep(a96) protocol. The reagent plates of the kit needed the automated DNA extraction pre-filled with all reagents.

Verification of the DNA Extraction

Agarose gel electrophoresis was used to confirm the extracted DNA. 10 µl of each eluate and 7 µl of the DNA ladder was applied to a 0.8 % TBE gel, containing ethidium bromide; 5 µl of ethidium bromide for 100 ml of agarose gel. A horizontal gel electrophoresis system (Analytik Jena AG) was used to complete electrophoretic separation through the constant application of 127 mA for ideal running conditions.

The consequential DNA bands were visualized with the use of UV light. A spectrophotometer (ScanDrop® 250, Analytik Jena AG) was used to determine both the yield and quality of the DNA, and 4 µl of each eluate were 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

Automated extraction of genomic DNA (gDNA) from 10 different whole blood samples (3 ml) was completed by combining smart Blood DNA Midi prep (a96) with CyBio® FeliX. The quality and yield of the isolated DNA were determined using the agarose gel electrophoresis, as seen in Figure 3, and spectrophotometric measurements, as seen in Table 1.

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 explains the purity (A₂₆₀:A₂₈₀ and A₂₆₀:A₂₃₀) and concentration of the extracted DNA. The measurements of purity and concentration were completed using Analytik Jena AG’s ScanDrop® 250. In the interest of the A₂₆₀:A₂₈₀ and A₂₆₀:A₂₃₀ evaluation, the extracted DNA shows brilliant quality. A high yield of high molecular weight DNA is enabled through the extraction method. Additionally, the samples were randomly distributed on the plate so that comparability and reproducibility of sample preparation inside the reaction plate could be demonstrated.

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

On the basis of a modular system, the degree of automation can freely be configured by the customer. The flexible automation solution contributes to better reproducibility of the results and increases the efficiency of laboratory processes.

Automatic extraction of DNA for downstream processes leads to easy, parallel preparation of samples with minimum effort and increased consistency. In addition, the CyBio® FeliX can be used for other downstream routine liquid handling tasks.

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 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: Feb 18, 2020 at 5:09 AM

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