Response to Bortolotti et al. 2012—a re‐evaluation of our polymerase chain reaction‐restriction fragment length polymorphism genotyping method

Recently, Bortolotti et al. described a real time single nucleotide polymorphism (SNP) genotyping assay for the HLA-G +3142 C>G polymorphism (1).In their study, 221 DNA samples from healthy subjects were genotyped by three different methods, including polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP), realtime PCR and DNA sequencing.The PCR-RFLP protocol was originally described by our research group and has been applied in different studies (2, 3).To our surprise, Bortolotti et al. reported that 19% of the individuals genotyped as heterozygous by PCR-RFLP were identified as homozygous for the G allele (GG) by real-time PCR and sequencing methods.The authors explained this discrepancy by high rates of partial digestion in the PCR-RFLP assay and, therefore, suggested that the PCR-RFLP technique was unreliable.In order to check for the reliability of the PCR-RFLP method designed by our group, the replicability of genotyping results for the HLA-G +3142 C>G polymorphism between this same PCR-RFLP methodology and DNA sequencing was verified.For that, we analyzed 289 DNA samples from critically ill subjects who were previously sequenced for the whole 3 UTR (untranslated region) of the HLA-G gene (4).These patients were genotyped in this study by the PCR-RFLP method described by our group and the genotyping results from the two methods were compared.The amplification of the HLA-G +3142 C>G polymorphism was performed as described in the study from Cordero et al. (2): 50 ηg-250 ηg of genomic DNA were added to a final volume of 25 μl, with final concentrations as follows: PCR buffer 1×, 2.0 mM MgCl 2 , 0.2 mM of each dNTP, 1.0 U of Taq DNA polymerase and 10 pmol of each primer (GmiR-NA_F 5 -CATGCTGAACTGCATTCCTTCC-3 and GmiR-NA_R 5 -CTGGTGGGACAAGGTTCTACTG-3 ).PCR was performed in a Techne TC-412 thermal cycler (Bibby Scientific Ltd., Stone, UK).Samples were submitted to 94 • C for 5 min, 32 cycles of 30 s at 94 • C, 30 s at 65.5 • C and 60 s at 72 • C, followed by a final extension step of 5 min at 72 • C. The amplified products of 406 bp were then submitted to digestion with 3 U of the BaeGI restriction enzyme.Specifically, 8.7 μl of amplified PCR products were mixed with 0.3 μl of BaeGI (10,000 U/ml) and 1.0 μl of NEBuffer 1 10× (New England