A series of papers from Prof. Walter Doerfler’s laboratory (Doerfler, 2000; Doerfler et al., 1997; Hohlweg and Doerfler, 2001; Schubbert et al., 1994; Schubbert et al., 1997; Schubbert et al., 1998) have addressed questions on the fate of ingested DNA. In preliminary work (Schubbert et al., 1994), mice were fed circular M13 bacteriophage DNA (~ 7.2 Kb) and scientists were able to detect small DNA fragments in certain organs and tissues. These fragments were mostly 200 - 400 bp in size, although up to 1.7 Kb fragments were detected in the feces and up to 500 bp fragments were detectable in the blood. These DNA fragments were detectable within 2-7 hours after feeding. The sum of all of the DNA fragments recovered from all tissues and feces could account for 2-4% of the total M13 fed to the mice, with only 0.01% detectable in the blood. Therefore, 96-98% of the ingested DNA was likely digested quickly and completely to very small and undetectable pieces. Furthermore, in vitro incubation of intact M13 DNA in blood demonstrated complete elimination within 6 hours. The results from this pioneering work were consistent with the general understanding that DNA is a normal component in food and subjected to extensive degradation during digestion. The authors stated “The implication that a random mixture of DNA including gene fragments or intact genes of animal, plant or microbial origin should have been constantly excreted by innumerable organisms over millennia does not appear startling given the complexities of evolution. This barrage of linear DNA fragments, i.e. of recombinationally highly active DNA fragments in Nature should mitigate any concerns that one might have had in the past about biological consequences of experiments carried out with recombinant DNA over the course of the past two decennia.” Earlier research46 complements these reports on studies on the fate of free DNA demonstrated complete and quick digestion in the intestine.

Klotz and Einspanier (1988) published that the gene responsible for glyphosate tolerance in soybeans was not detectable by PCR followed by Southern blot in either the blood of the cow or the milk from these animals. This publication showed that the highly sensitive method of PCR followed by a Southern was able to detect a small fragment of a highly abundant endogenous chloroplast gene in blood lymphocytes but not milk. Very recently, Einspanier’s laboratory has published data from a study in which dairy cows, beef steers and broiler chickens were fed either conventional maize grain or Bt corn (Einspanier et al., 2001; Flachowski et al., 2000). The investigators evaluated two DNA detection technologies [standard Polymerase Chain Reaction (PCR) and Light Cycler “real time” PCR]. Although Light Cycler PCR showed advantages for detecting Bt-maize in feed, this technique did not provide additional sensitivity beyond standard PCR methods for animal tissue samples. The presence of even a small portion of the coding region of the Bt gene was not detectable by either standard PCR or Light Cycler PCR in any samples from the cows, steers or chickens fed Bt corn. Similar to their previous report, using standard PCR technology, a small portion of the coding region of a highly abundant chloroplast gene (tRNAleu) was detectable in lymphocytes of dairy cows and in muscle, liver, spleen and kidneys of chicken, but not in dairy milk, or any tissue samples from steers. It is important to note that plastid genome copy number per cell varies depending on tissue-type, ranging from ~500 to 10,000 copies in roots and leaves, respectively (Bendich, 1987). Therefore, the copy number of plastid genes is orders of magnitude higher than a transgene in a biotech product, which typically has only one copy present per haploid genome. In addition, plastid gene sequences are also present in high numbers in the nuclear genome, with sometimes >100 copies of some sequences being observed (Ayliffe et al., 1998), such that the nuclear copies of plastid genes are an additional source of positive PCR signals. As a consequence, the high copy number of plastid genes and their subcellular localization within organelles could explain detection of these endogenous genes while transgenic DNA fragments are undetected to date.

Khumnirdpetch et al. (2001) attempted to detect transgenic DNA in broiler chickens. Broiler chickens were maintained by commercial standards and fed diets containing meal from either conventional or glyphosate tolerant soybeans from birth to seven weeks of age. Samples (meat, skin, duodenum and liver) were isolated from the birds at 1, 3, 5 and 7 weeks. Real-time PCR was used to test for the transgenic DNA in the various samples. PCR results of the broiler samples taken over this entire seven-week feeding period were all negative. The authors speculated that the negative detection results suggest that the transgenic DNA in glyphosate tolerant soybean meal has been fully degraded in the digestive tract of the broilers.

Weber and Richert (2001) also reported data on PCR studies attempting to detect both the Bt gene and an endogenous corn gene in DNA extracted from 24 pork loin samples (12 fed Bt corn and 12 fed a control conventional corn). PCR, followed by Southern blot analysis for ~200 bp fragments of the cry1Ab and shrunken-2 (sh-2) were uniformly negative. The sh-2 gene is an endogenous single-copy corn gene. By comparison, an endogenous swine gene (pre-prolactin) was readily detected in all pork loin samples, and spiking corn DNA into the extracted swine DNA also yielded positive results, indicating that the DNA quality and PCR conditions were both favorable for detection of DNA fragments, had they been present in the original samples. The PCR assay coupled with Southern blot was shown to have a limit of detection of approximately 1 to 2.5 pg of target DNA per 1 mg of input DNA, or approximately 1 genome equivalent of the target gene per PCR, the theoretical limit of assay sensitivity.

DNA degradation during the digestive process has been documented from mouse feeding studies with M13 phage DNA and recently reviewed (Doerfler, 2000). From studies feeding purified M13 phage DNA to mice, it was observed that up to approximately 0.1% of that ingested DNA could be detected in their blood (Schubbert et al., 1998; Schubbert et al., 1997). This extremely high level of DNA observed in the circulation is most likely owing to unique features of this circular, non-methylated phage DNA. Using the M13 data from mice, however, a calculation can be performed to predict the theoretical level of transgenic DNA that might be present in animal tissues, assuming uniform tissue distribution of that DNA in the farm animal. Basing uptake of transgenic DNA in farm animals on the mouse M13 phage data, it can be estimated that approximately 0.002 fg of transgenic DNA (1 femtogram equals one-trillionth of a mg, or 10-15 of a gram) might be present per mg of muscle tissue in the farm animal. No transgenic DNA has been detected in meat, milk or eggs from farm animals fed biotech products. These results are consistent with the knowledge that there are extremely small amounts of transgenic DNA in plants improved through biotechnology biotechnology (<0.004% of the total plant DNA).

However, it is important to remember that even if transgenic DNA is detected by a future study, scientific evidence and opinion concludes that ingested transgenic DNA would not be any different from ingestion of DNA already in foods, which is deemed safe. The safety of ingested DNA cannot only be derived from the long natural history of animal and human consumption of DNA, but it is also significant that, as would be expected because of digestive processes, no intact genes, only relatively small fragments, have yet been detected in animal tissues, regardless of the gene’s abundance. Instead, in the published reports describing detection of DNA from ingested plants in animal tissues, only small portions of the entire coding region of these highly abundant chloroplast genes were found (Einspanier et al., 2001; Klotz and Einspanier, 1998). Furthermore, only samples from a fraction of the total number of tested animals are yielding positive detections for these highly abundant gene fragments, suggesting that most of the individual animals are degrading ingested DNA to levels below the most sensitive PCR detection limits. Therefore, the likelihood that a transgenic gene or fragments is absorbed to any significant degree following digestion remains extremely low, especially when the relatively low levels of the transgenic DNA per cell is also considered when compared to the highly abundant endogenous plastid genes.