It is indeed interesing to witness real scientic debate is alive and well in crusty scientific journals:
Molecular Ecology, 2009 News and Views
COMMENT
Insufficient evidence for the discovery of transgenes in Mexican landraces
BERND SCHOEL and JOHN FAGAN
Genetic ID NA, Inc, SO4 N 4th Street, Fairfield, Iowa 52556, USA
The authors claim either sampling effects or reporting of false negatives as the most likely source of differing detection results between their study and that of Ortiz-Garcı´a et al. (2005). Our interpretation leads to the conclusion that Pin˜eyro-Nelson et al. (2009) essentially came up with negative results in their survey of Oaxaca for transgenic maize.
Although sample 5 appears to be positive, it is hard to conclude from the provided data whether this is a true positive result as the authors provided neither confirmatory Southern blot data nor information regarding the specific corn event. This interpretation is consistent with the conclusions reported by Ortiz-Garcı´a et al. (2005). We contend that the sample number was too small in both the study (Ortiz-Garcı´a et al. and Pin˜eyro-Nelson et al.) and that sampling was not representative of the total Oxacan maize population. Therefore, our conclusion from both publications on this topic is that results obtained to date are not sufficient to ascertain whether introgression of transgenic traits into the Mexican maize population has or has not taken place.
References include
Ortiz-Garcıa S, Ezcurra E, Schoel B et al. (2005) Absence of detectable transgenes in local landraces of maize in Oaxaca, Mexico.
Proceedings of the National Academy of Sciences, 102, 12338–12343.
Pineyro-Nelson A, van Heerwaarden J, Perales HR et al. (2009) Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations. Molecular Ecology, 18, 750–761.
Response
Molecular Ecology, 2009 News and Views
REPLY
Resolution of the Mexican transgene detection controversy: error sources and scientific practice in commercial and ecological contextsA. PIN˜ EYRO-NELSON,* J . VAN HEERWAARDEN,† H. R. PERALES,‡J . A. SERRATOS-HERNA´ NDEZ,§ A. RANGEL,–M. B. HUFFORD,**, P. GEPTS,** A. GARAYARROYO,*
R. RIVERA-BUSTAMANTE– and E. R. A ´ LVAREZ-BUYLLA*
Bernd Schoel and John Fagan (Vice-President and Founder ⁄ CEO, respectively, of Genetic ID, henceforth BS&JF) criticize and dismiss our recent publication in Molecular Ecology by focusing on our use of the Polymerase Chain Reaction (PCR) to detect specific DNA sequences. They raise important questions about the standards required to use PCR in various environmental conditions, pointing to the well-known fact that this delicate method may lead an unskilled operator to false results. They further suggest that our observations of transgenic DNA sequences in Mexican landrace maize should be attributed to false positives, i.e. a type I error. After considering their challenge and reviewing the evidence, we find their arguments seriously
lacking in substance, and their practice permissive of false negatives, a type II error.
We seem to have attracted BS&JF’s attention because, in an effort to corroborate our own results, we utilized the services of Genetic ID as full-paying customers. We established that Genetic ID failed on occasion to detect positive blind samples, which should not be surprising given the known vagaries of the PCR method. Yet for BS&JF this detection failure is not a
factual possibility; instead, to explain our observations they would have us both (i) contaminating our samples and (ii) lying about the origin and nature of our materials. Specifically, BS&JF state:
1 ‘We contend that results such as these are incorrectly interpreted as positive and are more likely to be indicative of contamination in the laboratory.’ and
2 ‘We would argue that the leaf sample provided by the authors did not contain the claimed NK603 event and, furthermore, does not contain material from any commercialized
transgenic single plant.’
Other charges include an implication that we used false evidence and ⁄ or withheld inconvenient data (BS&JF, p.5, lines 5–11) to reach our conclusions.
All of these are indeed very serious challenges to our technical capacity and expertise, as well as our professional and personal integrity.
PCR contamination or false negatives (type II error)?BS&JF declare their suspicion that all of our PCR positive results arose from systematic contamination. They note the presence of bands in the PCR gels that are weaker than they would expect for a ‘100% (homozygous) or 50% (heterozygous) GMO level’, the only evidence that they would take as a positive result. Such a view is based on the unwarranted expectation
that an end-point PCR could be used as a quantitative method. In our experience and that of other independent laboratories, the PCR amplification of transgenic sequences in landrace maize backgrounds tends to produce relatively faint bands of variable intensity in end-point reactions visualized on agarose gels, which so far has been the standard approach in the field (Quist &
Chapela 2001; Alvarez- Morales 2002; Pin˜ eyro-Nelson et al. 2009). Genetic ID’s own gels (their standard to screen-out ‘negatives’) show this kind of variability, even for repeats of a single sample in a single assay, or for different assays performed for the same sample at different times [see Fig. S1 (Supporting information)]...
...There is evidence arguing against BS&JF’s PCR contamination hypothesis. We observe, for example, that the presence of positive bands in our samples is neither randomly nor homogeneously distributed as would be required by such a hypothesis. Specifically, at the inception of our study, maize ears were collected, seeds were subdivided from each ear and distributed independently to our separate laboratories (RR in Irapuato and EAB in Mexico
City) by an outside researcher (S. Ortı´z-Garcı´a, a co-author with B.S. in Ortı´z-Garcı´a et al. 2005). Maintaining each laboratory in complete isolation from the other through double-blind coding, seeds from these subsamples were germinated, emergent leaf tissue lyophilized in facilities free of cloning or PCR products, extracted and PCR-amplified, after which we compared all results for congruence. As explicitly described in our original study, we took a highly
conservative position before we would call a positive sample: samples were never scored as positive unless we had at least two repeated confirmatory results in each separate laboratory based on independent DNA extractions and amplifications.
Under these circumstances, the laboratory contamination implied by BS&JF should be expected to either appear in all samples or to be randomly distributed among families within laboratories, with a possible differentiation between the two laboratories reflecting their differing patterns of contamination. None of these scenarios occurred. Families and ⁄ or localitiesconsistently appeared with positive individuals in both laboratories while others consistently failed to show positives. We have now subjected all our results to a statistical analysis showing that the distribution pattern of positive samples among seed families or localities is indistinguishable between the two independent laboratories; i.e., overall, the frequency of positives among families matches across laboratories. The probability of this pattern emerging from a contamination source is <0.001.>
There are good reasons to believe that such limited focus may place Genetic ID’s methods at a relative disadvantage for detecting transgenic DNA sequences in landrace maize. Using real-time PCR, we found that there are significant differences when comparing a hybrid transgenic commercial line against a landrace sample in the relative amplification of an internal control, a zein gene, included in the TaqMan(R) kit for the quantification of the 35S CaMV promoter sequence (see Fig. 1.)... 1 We have already pointed out the expectation of much higher levels of molecular diversity in Mexican landrace samples with a diverse genetic background compared with hybrid, commercial varieties (Pin˜eyro-Nelson et al.2009). Significant genome size variation among landraces has been reported (1700 to 3300 megabases; see comment in Walbot 2008), while lack of genetic colinearity and pervasive gene duplication have been described (Fu & Dooner 2002; Wang & Dooner 2006).
We stand by our expectation that such diversity could cause inefficiencies and variability in PCR results stemming from direct or indirect molecular effects on any of the components and conditions of PCR assays. In these conditions, a protocol with no flexibility for careful observation and follow-up of bands that are less than optimal would create ample opportunity for false negatives.
2 BS&JF dismiss any discussion of PCR inconsistencies by vaguely invoking an undefined and unaccountable protocol, thus: ‘[Genetic ID] includes at several points in its analytical procedures controls that would detect the kinds of problems cited by the authors and therefore ensure accurate reporting of results. For example, PCR inhibition tests are routinely conducted to rule out the presence of compounds ‘metabolites’) that could interfere with PCR amplification.’ Of particular interest is their claim of a standard, routine test for inhibition of the PCR assay, which should stand for any and all sources of inhibition possible from commercial and landrace materials that have a wide range of, for example, phenolic compounds in their constitution (Arnason et al. 1994); no details are given about the specific sequences used in such tests,gene dosage or specific genetic behaviour. Our own experimental routine shows this facile dismissal of the inhibition problem to be fallacious. Specifically, we showed as part of our careful method evaluation that there are indeed differences between commercial, hybrid maize varieties and landrace materials as far as their PCR performance is concerned.
3 In their critique, BS&JF deride our expectation of sequence diversity in our target sequences by claiming that such an expectation violates ‘the known and accepted norms of genetics’ (Schoel & Fagan, p. 3). BS&JF’s sole source of support is a general evaluation of the average rate of spontaneous mutation across broad taxonomic groups (Drake et al. 1998). This approach fails to recognize site-specific differences in mutation rates, especially well known in transgenic constructs where, for example, the borders of the transgenic construct are prone to sequence variation (Matsuoka et al.2002). Maize itself has highly variable mutation rates at different loci, ranging from <0.1>Selected References
Fu H, Dooner HK (2002) Intraspecific violation of genetic colinearity and its implications in maize. Proceedings of the National Academyof Sciences USA, 99, 9573–9578.
Drake JW, Charlesworth B, Charlesworth D, Crow JF (1998) Rates of spontaneous mutation. Genetics, 148, 1667–1686. Matsuoka T, Kuribara H, Takubo K et al. (2002) Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays). Journal of Agricultural and Food Chemistry, 50, 2100–2109.
Pin˜eyro-Nelson A, van Heerwaarden J, Perales HR et al. (2009) Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations. Molecular Ecology, 18, 750–761.
Walbot V (2008) Meeting report: maize genome in motion. Genome Biology, 9, 303. doi:10.1186/gb-2008-9-4-303)
Wang Q, Dooner HK (2006) Remarkable variation in maize genome structure inferred from haplotype diversity at the bz locus. Proceedings of the National Academy of Sciences USA, 103, 47.
Labels: Environmental management, Ethics, Genetics, Stakeholder disagreements
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