Two hundred microliters of each blood sample were homogenized
in a 1.5-ml microcentrifuge tube using disposable
polypropylene pestles. The extractions
were performed with the Nucleospin Tissue kit . Next, 50 ul of extracted DNA were eluted with
5 mM Tris–HCl (pH 8.5), and each sample (5 ul) was subsequently
used for PCR amplification to generate copies of the DNA templates
for pyrosequencing..