Introduction

This protocol describes a method to extract genomic DNA (gDNA) from human blood collected on Whatman’s Flinders Technology Associates (FTA) cards. The blood was collected in EDTA K2 solution and applied to the FTA cards upon arrival in the laboratory. The blood cells were counted before application to the FTA cards to assess the efficiency of this method. Optimal results were obtained using 10 discs of 3 mm diameter each, pre-lysed for one hour. The discs were loaded into the QIAGEN EZ2 Connect automation device using the tissue protocol for 15 minutes to complete purification. Different numbers of discs were tested and results showed 10 discs was the maximum which can be used whilst still obtaining consistent DNA recovery. The Ligation Sequencing Kit was used to generate sequencing libraries from the extracted DNA, and the libraries were sequenced on the PromethION device.

The QIAGEN MagAttract kit may also be a suitable alternative to extract the gDNA as it is based on the same principle of magnetic beads, but this method has not been tested.

Materials

• Blood collected on FTA cards
• 3 mm puncher
• Cutting mat
• Tweezer
• Thermomixer, or incubator with heating capacity of 56°C and agitation capacity of 900 rpm
• 2 ml DNA LoBind Eppendorf tubes
• QIAGEN EZ2 Connect automation device
• EZ 1 & 2 DNA Tissue Kit

Method

1. Apply the blood to the FTA card and allow the sample to completely dry.
2. Place the FTA card on the cutting mat and cut out 10 discs using a 3 mm puncher.
3. Using tweezers, transfer the 10 discs to a 2 ml DNA LoBind Eppendorf tube.
4. Add 290 µl of G2 buffer and 10 µl of proteinase K.
5. Incubate the sample on a thermomixer, or equivalent, for 1 hour at 56°C and 900 rpm.
6. Remove the tube from the thermomixer and transfer the lysate to the sample tube provided in the kit by pipetting. Transfer as much lysate as possible, being careful not to transfer any FTA discs as these can block the instrument's tips.
7. Set up the automation device according to the manufacturer’s instructions, select the tissues protocol and set the elution volume to 50 µl.

Results

Typical protocol yield: 200-450 ng

The DNA library for sequencing was prepared using the Ligation Sequencing Kit.

Figure 1. Read length analysis of the extracted gDNA from human blood samples.

Figure 2. Output analysis of the extracted gDNA from human blood samples.