Blood samples from wild birds, 30 males and 50 females of Scopoli’s Shearwater (Calonectris diomedea), 20 males and 25 females of European Storm Petrel (Hydrobates pelagicus melitensis), 3 males and 3 females of Yelkouan Shearwater (Puffinus yelkouan), previously sexed by PCR with standard primers (CHD2550 and CHD2718) have been employed to test and validate the method. Further C. diomedea samples were employed (1200 of blood and 130 of feathers), among which males and females resulted nearly equally represented.
Samples were collected during the breeding seasons. Freshly sampled blood (one single drop of 1–3 μL or, alternatively, a few mm2 of blood-spotted filter paper) was added to 50 μL of lysis solution (200 mM KOH, 20 mM Na2EDTA, 0.25% Triton X-100); in fresh blood, complete lysis occurs within a few seconds, and it results in sudden increase of the solution viscosity, due to free DNA. However, further incubation at ambient temperature for 1–2 min after lysis improves DNA and protein denaturation and solubilization (some blood proteins, such as proteases, are known to inhibit DNA amplification), as well as the dissociation of DNA/proteins complexes.
Dry blood stains absorbed onto filter paper (air-dried and stored up to several months at room temperature) were incubated in lysis buffer at 70–80 °C for 10 min or until the blood appeared dissolved (alternatively, incubation can be carried out overnight at room temperature); feathers (one per sample) require harsher conditions and were incubated at 98 °C for 5 min or, alternatively, at 80 °C for 15 min, followed by 1 min at 98 °C.
After a brief, thorough mixing, six volumes of a BTB neutralization solution (50 mM Tris-HCl, 0.0025% bromothymol blue) were added and the samples were briefly mixed. This makes the use of micropipettes not strictly necessary, and both solutions can be measured as drops (six drops of neutralization solution per drop of lysis solution). The pH indicator BTB was previously tested for its compatibility with amplification reactions and was included to check samples for proper pH values, compatible with downstream applications. In fact, the dye turns from yellow to blue/green around pH values of 7.6 (Figure 1 A), the correct pH is easily monitored by the color of the solution (Figure 1 B) and the resulting buffer system ensures suitable pH values even if slightly different volumes are added.
Samples were collected during the breeding seasons. Freshly sampled blood (one single drop of 1–3 μL or, alternatively, a few mm2 of blood-spotted filter paper) was added to 50 μL of lysis solution (200 mM KOH, 20 mM Na2EDTA, 0.25% Triton X-100); in fresh blood, complete lysis occurs within a few seconds, and it results in sudden increase of the solution viscosity, due to free DNA. However, further incubation at ambient temperature for 1–2 min after lysis improves DNA and protein denaturation and solubilization (some blood proteins, such as proteases, are known to inhibit DNA amplification), as well as the dissociation of DNA/proteins complexes.
Dry blood stains absorbed onto filter paper (air-dried and stored up to several months at room temperature) were incubated in lysis buffer at 70–80 °C for 10 min or until the blood appeared dissolved (alternatively, incubation can be carried out overnight at room temperature); feathers (one per sample) require harsher conditions and were incubated at 98 °C for 5 min or, alternatively, at 80 °C for 15 min, followed by 1 min at 98 °C.
After a brief, thorough mixing, six volumes of a BTB neutralization solution (50 mM Tris-HCl, 0.0025% bromothymol blue) were added and the samples were briefly mixed. This makes the use of micropipettes not strictly necessary, and both solutions can be measured as drops (six drops of neutralization solution per drop of lysis solution). The pH indicator BTB was previously tested for its compatibility with amplification reactions and was included to check samples for proper pH values, compatible with downstream applications. In fact, the dye turns from yellow to blue/green around pH values of 7.6 (
Free full text: Click here
