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DNA Extraction Methods For Large Blood Volumes

Introduction

DNA extraction is a key stage in molecular genetic testing. Automation of this process has some important benefits, including increased throughput, more consistent and reproducible processing and improved sample tracking.

Several manufacturers have produced different platforms, with different extraction chemistries like e.g. paramagnetic beads, salt extraction, filter systems, etc. However, the extraction of large blood volumes (>1 ml) is still a challenge, where only a few systems are available.

We will first evaluate the Magnetic Separation Module I, produced by Chemagen. This instrument is built around the paramagnetic beads chemistry. Blood samples, beads and a series of wash solutions are loaded on the horizontal track together with empty tubes to collect the re-suspended DNA samples. The track is able to move backwards and forwards to locate the appropriate position under the robotic head (fig 1). When the electromagnet is turned off, the separation head can be used to vortex the solutions with the rotating rods. If switched on, the paramagnetic tips of the rods are activated, which allows the beads to be picked up and transferred from tube to tube (fig 2).

The instrument is controlled by a simple software program on a PC. Extractions are possible for both small (96 well plate format for 200µl) and large volumes (3x4 rod array for 3-7ml) of blood (fig 3).

Fig 1: the Chemagen Magnetic Separator Module I
Fig 2: When the electromagnet is turned off, the separation head can be used to vortex the solutions with the rotating rods. If switched on, the paramagnetic tips of the rods are activated, which allows the beads to be picked up and transferred from tube to tube
Fig 3: a) 96 well plate format robotic head; b) 3x4 rod array robotic head

Aim

The final aim is to generate a complete technical validation report of the Chemagen Magnetic Separator Module I platform in combination with a Perkin Elmer Multiprobe robot for large blood volumes.
This will help other laboratories to implement and validate this technology in-house. Furthermore this report can serve as a template for validation of other platforms, which can be evaluated in other accredited laboratories.
The validation report will describe the Chemagen technology and include a detailed evaluation of the physico-chemical qualities of the extracted DNA.
In addition, the performance of the extracted DNA in downstream diagnostic tests will be evaluated.

Work flow

The starting point for this validation:

  • at least 180-200 blood samples will be extracted (15-20 runs)
  • a fixed volume of blood (5ml) will be used
  • the anticoagulant will be EDTA
  • the blood samples will be "fresh" (not older than two weeks) and stored at room temperature until extraction
  • no blood samples from patients with blood-related diseases, e.g. leukaemia, will be included

The parameters to be tested:

  • Contamination: 24 blood samples (spread over 4 independent runs) will be alternated with blank samples (1xPBS) to evaluate possible cross-contamination during the extraction procedure.
  • DNA concentration: the Nanodrop and the Victor spectrophotometers will be used to measure the DNA concentration.
  • DNA yield: will be expressed as amount of DNA per ml of blood and as amount of DNA per million white blood cells. The latter might give a more realistic picture.
  • DNA purity: 260/280nm and 260/230nm ratios will be measured, using the Nanodrop.
  • DNA quality: each DNA sample will undergo a multiplex drop-out PCR (van Dongen et al. 2003), to distinguish good and bad quality samples.
  • DNA degradation: 30 DNA samples will be randomly selected and loaded on a 0.8% agarose gel in order to check for degradation of the DNA samples.
  • DNA stability: 8 DNA samples will be randomly selected from independent runs and stored under different conditions, i.e. at a high temperature (e.g. 60°C), at room temperature and at -20°C to undergo some freeze-thaw cycles. These aliquots will be weekly tested for DNA quality and degradation over a period of at least two months.
  • DNA yield repeatability: the same blood sample will be extracted 3 times in the same extraction run and this will be done for 4 different blood samples.
  • DNA yield reproducibility: the same blood sample will be extracted in 3 different extraction runs on 3 different days by 3 different operators. Again 4 different blood samples will be used.

This initial validation of the Chemagen platform can be extended with:

  • extraction for small blood volumes (200µl in 96 well plate format)
  • a range of large blood volumes (1-10 ml)
  • an alternative anticoagulant, e.g. heparine, citrate
  • "older" (older than two weeks) blood samples
  • samples from patients with blood-related diseases
  • other sample types, e.g. placenta, saliva, CVS, ...

End result

The final outcome will be:

  • a complete validation report, discussing in detail the results of the evaluated parameters, which can be used as a model to validate other platforms.
  • a generic SOP to install and use the Chemagen Magnetic Separator Module I in any laboratory.

References:

  • National Genetics Reference Laboratory (Wessex), Technology Assessment report: Automated DNA extraction - Gentra Autopure LS, September 2004
  • National Genetics Reference Laboratory (Wessex), Technology Assessment report: Automated Extraction Methodologies, November 2004
  • van Dongen et al. (2003), Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936, Leukemia (2003) 17, 2257-2317

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