Method of the GOOD Assay

Ohgonucleotides for the PCR and primer extension reactions were synthesised and HPLC purified by MWG Biotech (Ebersberg, Germany). For the amplification of a fragment of the interleukin 6 gene primers 5 -CCTGGAGGGGAGATAGAGCTTCT-3 (forward) and 5 - 

CCCTAGTTGTGTCTTprG FiC-3 for the SNP 987 in the interleukin 6 gene were used as primers for the primer extension reaction. 

Charge tagging The amino functionality of the synthesised primers was used for attaching a positive charge tag (6-trimethylammoniumhexyryl-N-hydroxy-succinimidylester) according to Gut et al. [17] and Bartlett-Jones et al. [16]. The primers were dissolved in 1 % TE-buffer to 5(X) pmol/pl. 30 pi of this solution were mixed with 1.5 pi 2 M triethylammoniumhydrogencarbonate (pH 8,0) and 24 pi fresh 1% 6-trimethylammoniumhexyryl-N-hydroxy-succinintidylester solution. This 

Shrimp alkaline phosphate (SAP) digestion 0.5 pi (1 U/pl) of shrimp alkaline phosphatase were added to the PCR reaction and incubated for 1 h at 37°C. The enzyme was denatured for 10 min at 90°C. 

Primer extension 25 pmol of the charge-tagged primers were added together with 4 mM MgCl2, 0,2 mM MnCl2, 100 pM a-S-ddNTPs and 0.5 U Thermosequenase. The reaction volume was increased to 20 pi by the addition of water. An initial denaturing step 2 min at 95°C was done, followed by 35 cycles of 20 sec at 95°C, 1 min at 60°C and 30 sec at 72°C. 

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