pyrosequencing which utilizes a cascade of coupled enzymatic reactions to monitor dna synthesis the pyrosequencing method is described in u s pat nos 6 210 891 and 6 258 568 wherein the entire contents of both patents are hereby incorporated by reference and ronaghi et al 1998 a sequencing method based on real time pyrophosphate science 281 363 365 the entire contents of which are hereby incorporated by reference the pyrosequencing technique has the advantages of speed accuracy and parallel processing the 5 u2032 end of the reverse primer used in the pcr was biotinylated to facilitate amplicon capture and preparation for pyrosequencing directly from the pcr using streptavidin sephadex amersham biosciences uppsala sweden and the pyrosequencing vacuum prep tool biotage uppsala sweden for the pyrosequencing reaction a 0 5 nm concentration of the sequencing primer 5 u2032 tctgggtagtaccatcagt 3 u2032 seq id no 18 was used this sequencing primer possesses a unique sequence found within the amplicon upstream from the codon which encodes the amino acid corresponding to the amino acid at position 54 of the cyp51a protein a pyrosequencing 96ma system biotage uppsala sweden was programmed with 10 cycles of an agct dispensation order the resulting pyrosequencing data was analyzed with the psq 96ma version 2 1 software biotage uppsala sweden the best quality dna sequences resolved were used in subsequent analyses typically 19 to 24 bases of sequence were interpretable.