Quist and Chapela reply

In contrast with the well-established PCR method, i-PCR is an exploratory method that depends on interpretation and the availability of known sequences in databases such as GenBank. We acknowledge that our critics' assertion of the misidentification of sequences labelled with adh1 intron 1 and with bronze1 is valid.

The suggestion of mispriming in our i-PCR reaction is also warranted for sequences AF434756 and AF434759 (ref. 1). Significant homology with putative misamplifications is maintained across the length of these fragments, and the CaMV sequence was not recovered. However, this pattern is not found in our other i-PCR sequences. A revealing pattern of discontinuity is found at at least one end of five other sequences, indicating the integration junction between the transgenic DNA and the native host genome. Our critics choose not to recognize this feature in the majority of our i-PCR data. Partial homology with retrotransposon elements in maize is common in primers designed to amplify transposon-like sequences, and is not unique to our primers. Questions concerning the distortion of expected footprints at the DNA-integration junction certainly warrant future work.

The movement of transgenes into new populations and across generations is expected to result in diverse integration patterns2,3,4,5,6,7. Our findings are compatible with recent studies2,3,4,5,6 that characterize transgene/host DNA junctions where rearrangements include interspersion with host or unidentifiable DNA. As altered DNA species should also be an important focus of ecological research, we disagree with our critics who assume that only intact transgenes are worthy of attention in our study.

We agree that PCR-based methods are sensitive and therefore open to artefacts, but strongly disagree that the presence of these artefacts is unavoidable or uncontrollable. The consistent performance of our controls, as reported1, discounts beyond reasonable doubt the possibility of false positives in our results. Nevertheless, the high sensitivity of the PCR reaction has incited some critics to request a non-PCR-based method to confirm our main statement. To address these challenges, we evaluated the same samples from our original publication1 using DNA–DNA hybridization. The results of these experiments continue to support our primary statement.

Our analysis of Oaxacan maize is unique for several reasons. First, we wished to document changes that occur within diverse populations of landraces (rather than single varieties or lines), for which no markers, restriction-enzyme digestion maps or linkage analyses have been developed. Second, we could not have predicted which (or how many) specific transgenic constructs (or derivatives) were present in the samples that we analysed. Third, our samples of ground, pooled kernels from individual maize cobs do not represent individual genomes. All of these factors render the application of DNA-hybridization methods difficult. To minimize confusion in interpreting the multiplicity of bands that would have been created by Southern hybridization with our samples, we chose to use dot blotting for our experiments.

We extracted genomic DNA from dry maize kernels1. Standards containing varying amounts of transgenic material were prepared by mixing flour from our positive control (Bt1) and our historical negative control1. We blotted and immobilized 10–15 µg of DNA from each sample onto a nylon membrane using a Bio-Dot apparatus (Bio-Rad). We generated a horseradish peroxidase-labelled DNA probe from the same 220-base-pair fragment of the p-35S CaMV promoter that was amplified from our previously reported1 positive control (Bt1). Hybridization conditions were as follows: 56 °C, 6 ng ml−1 DNA probe, 1 hour. Washes were as follows: 3 × 5 min with 0.1 × SSC/0.1% SDS at 56 °C, followed by 3 × 5 min with 2 × SSC at room temperature. Loading homogeneity was confirmed by stripping and rehybridization of the experimental membrane with the 329-base-pair fragment from the maize-specific zein gene1. Probe labelling, hybridizations and detection were carried out using a North2South kit (Pierce Endogen), according to the manufacturer's specifications.

DNA from four of our six criollo landrace samples, and from the Diconsa sample, hybridized with our CaMV probe (Fig. 1). By using standardized mixtures of transgenic and non-transgenic maize, dot-blot hybridization suggests a ratio of transgenic to non-transgenic kernels in criollo cobs of the order of 1:100, as we had previously suggested1 and as was confirmed by Mexican government studies1. This DNA-hybridization study confirms our original detection of transgenic DNA integrated into the genomes of local landraces in Oaxaca.

Figure 1: DNA–DNA dot-blot hybridization between maize genomic DNA and a CaMV p-35S probe. Sample numbers coincide with those in ref. 1.
figure 1

Top row: 1, 100% transgenic; 2, 10% transgenic; 3, 5% transgenic; 4, 1% transgenic, 5, 0.5% transgenic; 6, historical maize negative control; 7, water negative control; 8, Diconsa sample K1. Bottom row: 1, criollo sample B1; 2, criollo sample B2; 3, criollo sample B3; 4, criollo sample A1; 5, criollo sample A2; 6, criollo sample A3; 7, Peru maize negative control P1; 8, water negative control.

Footnote 1