DraI NEB#R0129

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	A region in intron 6 of CYP2E1 (7632T>A) was amplified and digested with DraI (8U, New England BioLabs). Wild type CYP2E1 allele (*1A) has restriction sites for RsaI and DraI, but not for PstI. The presence of restriction sites yielded two fragments of 352 and 143 bp for RsaI restriction digest, 377 and 118 for PstI, and 268 and 306 for DraI restriction digest.

Protocol tips
A region in intron 6 of CYP2E1 (7632T>A) was amplified and digested with DraI (8U, New England BioLabs). Wild type CYP2E1 allele (*1A) has restriction sites for RsaI and DraI, but not for PstI. The presence of restriction sites yielded two fragments of 352 and 143 bp for RsaI restriction digest, 377 and 118 for PstI, and 268 and 306 for DraI restriction digest.

Publication protocol

Genomic DNA was extracted from a whole blood sample of each patient according to the Salting Out method, modified from Miller et al. (Miller et al. 1988). Polymorphisms in CYP2E1 were detected using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method (Molecular Probes, Invitrogen Detection Technologies, OR, USA). Amplification was performed in a PTC-100™ thermocycler (MJ Research Inc) and PCR details are described in Supplementary Table I. After amplification, the fragment was digested with RsaI (5U New England BioLabs) and PstI (4U, Invitrogen, Brazil) endonucleases, at positions -1053C>T and -1293G>C, respectively. A region in intron 6 of CYP2E1 (7632T>A) was amplified and digested with DraI (8U, New England BioLabs). Wild type CYP2E1 allele (*1A) has restriction sites for RsaI and DraI, but not for PstI. The presence of restriction sites yielded two fragments of 352 and 143 bp for RsaI restriction digest, 377 and 118 for PstI, and 268 and 306 for DraI restriction digest. Digested products were analyzed by electrophoresis in 2.5 % agarose gel and stained with ethidium bromide or GelRed™. Presence or absence of GSTM1 and GSTT1 were observed by performing two multiplex PCRs. NAT2 genotyping method was described by Possuelo et al. (Possuelo et al. 2008).

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