For samples applied on FTA®cards, 0.2 mm discs were cut from the card for PCR analysis. The number of discs to be examined were cut and placed together into a micro-centrifuge tube for washing. To avoid cross contamination between samples, an equivalent number of discs were taken from a blank filter paper after each sample. The discs were washed twice, for 15 minutes each, in 200 μl (for each disc) of Whatman FTA purification reagent. The discs were then washed twice for 15 minutes in 200 μl (for each disc) 1 mM TE buffer (10 m M Tris-HCL pH 8.0; 1 mM EDTA pH 8.0), were then transferred to PCR tubes and left to dry at room temperature for at least 90 minutes [21 ]. Ten discs from both whole blood and lysed blood FTA®card preparations were examined by PCR (each disc examined with a separate PCR reaction).
The DNA from a further 10 prepared discs from each FTA®card sample was eluted collectively by heating the discs for 30 minutes at 90°C in 60 μl of 5% (w/v) aqueous suspension of Chelex 100® resin (sodium form, 50-100 dry mesh, Sigma) [23 (link)]. For PCR, 5 μl of the eluate was added to 20 μl of the PCR master mix.
DNA extraction directly from field sampling was carried out according to the manufacturer using the ChargeSwitch® gDNA kit. The principle of this extraction method is the use of magnetic beads. At low pH, the magnetic beads have a positive charge that binds the negatively charged nucleic acid backbone of DNA. Proteins and other contaminants do not bind and are removed by washing. For the elution of the bound DNA, the charge of the magnetic beads was neutralised by raising the pH to 8.5 using a low salt elution buffer. The purified DNA was released into the elution buffer; the yield from 50 μl of blood was up to 2 μg. For PCR, 1 μl from the extract was used as the template.
The DNA from a further 10 prepared discs from each FTA®card sample was eluted collectively by heating the discs for 30 minutes at 90°C in 60 μl of 5% (w/v) aqueous suspension of Chelex 100® resin (sodium form, 50-100 dry mesh, Sigma) [23 (link)]. For PCR, 5 μl of the eluate was added to 20 μl of the PCR master mix.
DNA extraction directly from field sampling was carried out according to the manufacturer using the ChargeSwitch® gDNA kit. The principle of this extraction method is the use of magnetic beads. At low pH, the magnetic beads have a positive charge that binds the negatively charged nucleic acid backbone of DNA. Proteins and other contaminants do not bind and are removed by washing. For the elution of the bound DNA, the charge of the magnetic beads was neutralised by raising the pH to 8.5 using a low salt elution buffer. The purified DNA was released into the elution buffer; the yield from 50 μl of blood was up to 2 μg. For PCR, 1 μl from the extract was used as the template.
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