Qualitative and Quantitative Analysis of the Fate of DNA in the Digestive Tract of Mice

Objective: Qualitative and quantitative examination of DNA degradation during the digestion process in the mouse gut through PCR, qPCR and short tandem repeat (STR) analysis. Methods: Human blood leukocytes were gavage into the digestive tract of mice. GAPDH, TH01, TPOX and D7S820 genes in the contents of the stomach and small intestine were analyzed through PCR and qPCR at various time pre- and post-gavage. Through STR analysis, 21 human genomic DNA loci were analyzed. The half-life of DNA degradation, and the relationship between the average peak area and digestion time were determined. Results: The PCR results showed DNA bands at pre-gavage (0 min) and post-gavage (40, 80 and 120 min) from the mouse stomach contents, whereas no DNA bands from small intestinal chyme were observed after gavage. The qPCR results revealed signicant decrease in DNA concentrations during 40-120 minutes in mouse stomach after gavage. At 120 min, 85.62±8.10% of the DNA was degraded while the half-life of exogenous DNA degradation in mouse stomach was 70.50±5.46 min. At various time of digestion , almost no target genes were detected in the mouse small intestinal chyme. STR analysis showed a decrease in allele numbers with the advancement of bowl in the small intestine of mice. Conclusions: The degradation of exogenous DNA was higher in mouse stomach during rst two hours while almost complete degradation was noted in the small intestine of mice within 40 min.


Introduction
Deoxyribonucleic acid digestion and absorption in the gastrointestinal tract (GIT) has been a topic of great interest. Studying the fate of DNA in the digestive tract can be used to assess the risk associated with food and medicine entering the body. Vieira [1] has used PCR to detect human blood DNA in the digestive tract of Aedes aegypti mosquitoes, thus facilitating epidemiological investigation and forensic identi cation.
Dietary DNA in the digestive tract has been thought to be completely hydrolyzed by digestive enzymes and acids in the mouth and GIT. However, this assumption was made without analysis of the sources or the remaining DNA fragments; instead, it was based on measurement of the biochemical degradation of DNA into single base pairs [2,3] . Subsequently, DNA was observed to degrade more rapidly in the upper part of the gastrointestinal tract than in the lower part, and plasmid DNA extracted from the intestinal contents of rats 5 hours after gavage has been found to be biologically active [4] . However, Loretz has reported that pig gastric juice and intestinal uid almost completely degrade plasmid DNA within 1 hour [5] . Rabbit DNA has been found in the blood of two volunteers who ate 400-600 grams of cooked rabbit meat [6] . DNA fragments have been observed in chicken muscle, liver, spleen and kidney [7][8] .
Similarly, soybean DNA fragments have been detected in the GIT, tissues and other organs of pigs, sheep and cattle [9][10][11] . However, Walsh [12] has detected cry1Ab genes and proteins in the digestive uids but not the tissues (i.e., kidney, liver, muscle, heart or blood) of pigs fed Bt MON810 corn. Sattarzadeh et al. [13] reported the presence of the Nptll gene and Nos promoter sequences in the stomach, but not other tissues, in F-DT and M-LT rats. Trojan reported no detection of purple wheat genes in the blood of chickens, rats and carp fed purple wheat [14] . Nawaz et al. [15] found that food DNA can be digested into DNA fragments up to several hundreds of bp, as detected in the GIT. Various researchers have reported similar observations of dietary DNA not only in the GIT in humans or animals, but also in the blood, other tissues or even various organs. The fate of dietary nucleic acids in animals has long been an exciting and controversial research topic.
Digestion of nucleic acids is generally believed to occur in the intestines [16] , the main site of digestion and absorption of nutrients and nucleic acids, the latter of which are hydrolyzed by nuclease, phosphodiesterase, alkaline phosphatase and nucleosidase into oligonucleotide-single nucleotide-nonnucleoside bases in the intestine [3,15] . Gastric juice consists of pepsin and gastric acid. Pepsin's main function is protein digestion, but in recent years, Liu [17] found that pepsin in gastric juice can digest not only protein but also nucleic acids. The digestion of nucleic acids starts in the stomach, and various animal pepsins have different abilities to digest nucleic acids [18] . Most dietary DNA is in the form of histones, which form nucleosomes. The complex components of the diet may affect the digestion of DNA by pepsin. Zhang [19] has demonstrated that common food components, including proteins, carbohydrates, metal cations and polycationic compounds, affect the digestion of DNA through in vitro simulation studies. Therefore, the digestion of DNA from different sources in animals requires further analysis.
The fate of DNA in the GIT in animals could be used as a model to assess the risk and e cacy of drugs and foods entering the body. The fate of digested DNA and the degradation rates of DNA from various sources in the GIT in different animals have not been studied in detail. Most researchers have studied the degradation of DNA in the GIT of various animals either in extracted animal gastric and intestinal uids or through in vitro simulation models. In in vitro studies, many factors must be controlled, such as temperature, pH, ionic strength, enzyme concentration, GIT ora and epithelium. Mice have been used as a model to explore the dietary DNA degradation and digestion process in the mouse GIT at various time intervals. The DNA degradation rate has been quantitatively analyzed.
At present, study of dietary DNA traceability has been mainly based on the polymerase chain reaction (PCR) technique. The minimum ampli able fragment length is 70-100 bp [20][21] . Gene degradation into fragments below 70 bp remains to be further studied.
Short tandem repeat (STR) sequences are a series of small DNA fragments of 2-7 bp repeating approximately 10-60 times in the human genome. STR loci are highly polymorphic and widely distributed. The allelic fragment range is small, and multiple detections of multiple loci can be performed. Allele loss caused by small fragment dominant ampli cation can also be reduced through STR analysis.
The target fragment after PCR ampli ed is small; therefore, STR analysis is suitable for the detection of degraded DNA [22] .
This study human blood genomic DNA was chosen as an exogenous gene to infused into the digestive tract in mice and used PCR, qPCR and STR automatic analysis to qualitatively and quantitatively analyze the degradation of genes in human genomic DNA into small fragments in the digestive tract in mice over time, and to calculate the DNA degradation rate and half-life.

Materials And Methods
2.1 We con rm that all methods were carried out in accordance with ARRIVE guidelines and regulations in manuscript, and all experimental protocols were approved by the University Ethics Committee of Dalian Medical University, the ethics number is: AEE18036.

Experimental animals.
Kunming mice were obtained from the Experimental Animal Center of Dalian Medical University. The mice weighed approximately 17-30 g, and both sexes were included.
2.3 Observation and calculation of the small bowel advancement rate.

Preparation of nutritive semi-solid paste.
Previously described methods were followed [23] with slight modi cations. A total of 5 g sodium carboxymethylcellulose was dissolved in 125 mL distilled water. Then the following were added: 8 g milk powder, 4 g sugar, 4 g starch and 2 g activated carbon. The mixture was stirred gently, and 150 ml containing approximately 150 g of a black semisolid paste was prepared. The product was stored at 4°C and was brought to room temperature before gavage.

Gavage in mice.
After fasting for 24 hours, the mice were gavage with a nutritional semi-solid paste (0.8 ml) and water.

Mouse sacri ce and dissection.
Three mice per group were sacri ced by cervical dislocation at the following time intervals: 10, 20, 30, 40, 50, 60, 70, 80, 90, 120, 150, 180, 360, 420, 510, 540 or 570 min. Through incision of the abdominal cavity, the stomach and intestine were separated. The small intestine was measured from the pylorus to the ileocecal region (d1). The distance from the pylorus to the mid-point of the black semi-solid paste was measured (d2). The small bowel advancement rate was calculated as: 2.4 Qualitative analysis of target genes by PCR.

Preparation of the buffy coat layer.
A total of 35 ml of human EDAT whole blood was obtained from a healthy volunteer donor at Dalian Medical University and informed consent was obtained from her. Blood samples were centrifuged at 5000 g for 10 min. Plasma was discarded, and the buffy coat layer was carefully collected and stored at 4°C.
The results of the agarose gel electrophoresis were analyzed with a UVPC-80 gel imaging system (UCP Inc., San Jose, CA, USA).

Gavage and DNA extraction.
Nutritive semi-solid paste was prepared as described above, except with the addition of 2 g of activated carbon powder. A total of 0.4 ml semi-solid nutrient paste was mixed with the buffy coat (0.4 ml). The nutrient semi-solid paste was administered to mice by gavage. Mice were sacri ced by cervical dislocation at time intervals of 0, 40, 80, or 120 min. Through incision of the abdomen, an equal amount of stomach and small intestine content was collected in a 1.5 ml EP tube. DNA was extracted according to the kit instructions and analyzed through PCR. The results of agarose gel electrophoresis were analyzed with a UVPC-80 gel imaging system. According to the qPCR ampli cation principle, the PCR product increases exponentially. Assuming that the ampli cation e ciency was "E", and the initial quantity of template was "N0", then the amount of PCR product after "m" cycles was "Nm". Hence, according to formula; Nm = n 0 (1+E) m . The initial quantity of template at 0 minutes of gavage was recorded as "N0", after "Ct0" cycles, and reached the baseline "Nq" while the initial quantity of template at different times of gavage was recorded as "Nn", at "Ctn" cycles, and reached the baseline "Nq". Therefore, N0(1+E) Ct0 = Nn(1+E) Ctn . So, assumed that the ampli cation E ciency was 1. Then, according to the formula , the relative quantity of DNA in stomach of mice at different digestion times was calculated.

STR result analysis
The relative concentrations of degraded DNA, expressed as the mean peak area, was calculated by dividing the sum of the interpreted peak areas by the total peak number of the full STR pro le (Table 1). STR patterns (Figure 1) ampli ed from gastric content DNA, 0 min after gastric gavage were shown as an example to illustrate the calculation method of the average peak area. Linear regression analysis was performed. The slope (K) was calculated between the natural logarithm (ln) of the average peak area and the digestion time. The half-life of DNA degradation in the mice stomach was determined using the following formula [25] :

PCR results of exogenous DNA target genes.
The selected exogenous target genes GAPDH, TH01, TPOX and D7S820 were analyzed for homologous sequences. DNA bands were detected for human target genes, whereas no bands were detected for mouse genes (Figure 4).

PCR results of DNA degradation in the mouse GIT.
The results of PCR in Figure 5 revealed the target DNA bands for GAPDH, D7S820 (i.e., lanes A1, A2, B1, C1, and D1) in the stomach contents at various times (0-120 min) pre and post gavage. No DNA bands were observed in the small intestine contents (i.e., lanes B2, B3, C2, C3, D2 and D3) at 40-120 min after gavage, as shown in Figure 5. The TH01 and TPOX gene ampli cation results (Figure 6) showed the presence of the target DNA bands in lanes A1 and A2 (0 min; pre-gavage); lanes B1, C1, and D1 represent the stomach contents and showed clear bands at 40, 80 and 120 min post-gavage, respectively. No DNA bands (TH01 and TPOX) were observed in the chyme obtained after 40-120 min from the small intestine (i.e., lanes B2, B3, C2, C3, D2 and D3) at various times after gavage, as shown in Figure 6. 3.4 qPCR results of DNA degradation in the mouse GIT.
The GIT contents were also analyzed through qPCR for the target genes (i.e., GAPDH, TH01, TPOX and D7S820) pre-gavage (0 min) and at different times post-gavage (i.e., 40, 80 and 120 min). The four target genes were ampli ed, and each group was analyzed three times. Pre and post-gavage times and the Ct values of four target genes were plotted in Figure 7.
The relative quanti cation of the four targeted genes at different digestion time was calculated, as shown in Table 3 and Figure 8. Signi cant decrease in DNA relative concentration was observed at 40, 80, and 120 min respectively, of post-gavage as compare with pre-gavage (Table 3). At 120 min 85.62±8.10% decrease in concentration was noted as compared with 0 min of gavage. According to the linear analysis, the digestion time and exogenous DNA degradation in mouse stomach was shown in Figure 9. The halflife of DNA degradation in mouse stomach was 70.50±5.46 minutes.   3.5 STR results of DNA degradation in the mouse GIT.
The STR patterns of the DNA samples obtained from the mouse stomach at 0 min post-gavage are shown in Figure 1. We used the average peak area as the concentrations of degraded DNA at different digestion times in the mouse stomach, as shown in Figure 11A. The concentration of DNA decreased after digestion in the stomach in mice at 0 min. The concentration of DNA did not change signi cantly during 40-80 min and remained stable. After 120 min, the DNA concentration decreased signi cantly.
A linear regression was calculated with the natural logarithm of the average peak area versus digestion time, as shown in Figure 11B. The degradation kinetic model was obtained. According to the formula: T½=, the half-life of DNA degradation in the mouse stomach was 63.13 min.
Through STR, the average peak area and the total number of alleles ampli ed were measured in various parts of the mouse digestive tract, and were 38, 4 and 1, in the stomach, upper small intestine and lower small intestine, respectively, at 40 min post gavage ( Table 5). The results after 40 min were comparable, and over time, fewer alleles were ampli ed. The STR average peak area in different parts of the GIT decreased signi cantly with the advancement of bowel/chyme, as indicated by the decrease in the number of alleles detected in the stomach, upper and lower small intestine in mice.

Discussion
Food and medicine are vital to human survival. Hybrid food products are produced to feed humans and livestock. Recently nucleic acid treatment through parenteral or GIT administration has been reported [26] . The nucleic acid fate in the GIT tract has been unclear. A mouse model was developed to study the fate of DNA in the digestive tract in mice.
The advancement rate was found to be 20.27±2.60% at 10 min post gavage. The advancement in the movement of bowel/chyme was observed in the GIT in mice. At 180 to 360 min post-gavage, the mouse stomach was completely emptied, and the contents of the stomach had fully entered the small intestine.
This nding is consistent to those reported by Chul-Hyun Lim [27] , who used a 13 C octanoic acid breath test to measure gastric emptying times in mice. After 510 to 570 min of digestion, all contents had entered the cecum. This would provide understanding of the advancement of bowel movement.
Currently, the most commonly used DNA-based detection techniques are PCR and qPCR. qPCR, the most commonly used DNA quanti cation method, has the advantages of low pollution and high speci city [28] . As shown in Figures 5 and 6, clear bands in the mouse stomach were observed pre-gavage (0 min) and post-gavage (i.e., during 40-120 min). These results con rmed the presence of human leukocyte DNA in the stomach in mice after 120 min of digestion (>200 bp). There were no observed DNA bands for the four target genes in the contents of the small intestine in mice at 40, 80 and 120 min after gavage. The DNA had degraded into fragments of <77 bp in the small intestine in mice. Nawaz [15] found that food DNA can survive in the digestion process, and DNA fragments up to several hundred bp can be detected in the GIT. Same as the results of other scholars [9][10][11][12][13] , DNA fragments can clearly be detected in the GIT of animals; however, whether they can be detected in the blood and other tissues requires further experiments.
Anatomically, the mouse stomach consists of two regions-the non-glandular/fore-stomach and glandular stomach-which are separated by a limiting ridge [29] . Gastric juice consists of pepsin and gastric acid. Pepsin's main function is to digest protein, but in recent years, Liu [17] found that the pepsin in gastric juice can digest not only protein but also nucleic acid. The digestion of nucleic acid starts in the stomach, and various animal pepsins have different abilities to digest nucleic acid [18] . Most dietary DNA is in the form of histones, which form nucleosomes. The complex components of the diet may affect the digestion of DNA by pepsin. Zhang [19] demonstrated that common food components, including proteins, carbohydrates, metal cations and polycationic compounds, are closely associated with the digestion of DNA through in vitro simulation studies.
Ct values in the gastric contents of mice at 0, 40, 80 and 120 min respectively after gavage were measured through qPCR as shown in Figure 7. The DNA concentration decreased consistently from 0 to 120 min post gavage, and at 120 min, 85.62±8.10% decrease was noted as compared with 0 min ( Table   3). The half-life of DNA degradation in mouse stomach was 70.50±5.46 min (Figure 9). This indicated that the DNA concentration in the mice stomach decreased signi cantly. Wiedemann S [30] analyze rubisco and cry1Ab gene through real-time PCR and reported that the degradation was 20% of the initial value at 2 hours as compare with 0 hour. The degradation was 0.5% of the initial value (0 hour) after incubated for 48 hours in the rumen.
DNA degradation may be associated with mechanical aspects, gastric juice and microorganisms in the mice stomach. The DNA was not completely degraded in the stomach of mice, and remained >200 bp of DNA fragments. Protein and carbohydrate, the main components of food, do not affect DNA digestion at the concentrations recommended by the WHO (40:1 and 80:1). When the ratio of protein to DNA is >80:1, DNA digestion is inhibited [18] . Divalent cations (Ca2 + and Mg 2+ ) can result in greater DNA digestion than monovalent cations (Na + and K + ) [18] . The gavage included nutritive semi-solid paste and human white blood cells. The sodium carboxymethyl cellulose, starch and milk powder in the nutritive semi-solid paste resembled normal dietary components, thus potentially inhibiting DNA digestion. In addition, the structure of human leukocytes includes a cell membrane and nucleus, which may protect against DNA digestion.
According to Zhang, pepsin has a digestive effect toward nucleic acid, on the basis of in vitro simulation: pepsin can digest speci c sequences nucleic acids, such as 5´-AAG↓AA-3´ and CGA↓T [17] . The target genes TH01, TPOX and D7S820 have repetitive sequences rich in TCAT, GAAT and GATA, respectively.
Mouse pepsin may have a restriction enzymatic effect on these sequences, thus resulting in DNA degradation.
In qPCR method, when the Ct value is >35, the target gene is considered absent. In Table 4, from the ampli cation curve, the Ct value of the mouse gastric contents at 40 min was approximately 26 (<35).
The Ct values of the four target genes in the upper part of the small intestine of the mice at 40 min were all less than 35, thus indicating that the four target genes were present in very low amounts; in the lower part of the mouse small intestine at 40 min, only the GAPDH gene had a Ct value less than 35. These results indicated the presence of a small amount of GAPDH, whereas the Ct values of the other three target genes were >35, indicating the absence of the target genes. Similarly, the target genes were not detected in the DNA of the small intestine contents in mice at other times after gavage ( Figure 10). These results were consistent with the PCR results, indicating that DNA was further degraded into small fragments <77 bp in the small intestine by digestive enzymes and intestinal microorganisms. The DNA in human white blood cells was more easily degraded digested by the gastric juices in mice when it entered the intestines.
The STR technique was used to amplify small fragments of DNA. The average peak area (38 alleles; 77-446 bp) of 21 gene loci ampli ed by STR provides a good representation of DNA degradation [9] . The capillary zone electrophoresis-laser induced uorescence method can be used to determine the DNA concentrations in serum and plasma, and is as accurate and sensitive as the widely used real-time PCR method [31,32] .
The results obtained from plotting the average peak area and gavage time were consistent with the results of qPCR ( Figure 11A). According to the natural logarithm of the average peak area versus the digestion time, the half-life of DNA degradation in the mouse stomach was 63.13 min. This result was consistent with the half-life of DNA degradation calculated by qPCR method. After food enters the stomach, through mechanical digestion and chemical digestion [33] , part of the food bolus enters the intestines in the form of chyme. Our results showed that the rate of cellular DNA degradation in the mouse stomach was slow. STR map analysis of DNA in the small intestine in mice revealed that as the chyme advances in the small intestine, the number of human DNA alleles decreased (Table 5). qPCR and STR both clearly showed that human genomic DNA was markedly more degraded in the mouse intestine than the mouse stomach, whereas human target genes were degraded gradually in the mouse intestine with chyme advancement. Gene degradation times were also predicted through the STR method ( Figure  11B).
Liu [19] et al. reported that nucleic acids are digested in the stomach in blackhead sh and banded grouper, whereas the digestion of nucleic acids by bovine gastric enzymes was not observed. Different animals have varying ability to digest nucleic acids with pepsin. According to the experimental results of the current study, the mouse pepsin can be assumed to have a digestive effect toward human genomic DNA, thus, providing a potential reference for future experiments.
In recent years, no detailed research has performed a quantitative analysis of the degradation of DNA in the digestive tract in mice. Our ndings should contribute to future food and drug research, and risk assessment of genetically modi ed foods.

Conclusions And Recommendations
The degradation of exogenous DNA was higher in mouse stomach during rst two hours while almost complete degradation was noted in the small intestine of mice within 40 min.    Lane M : 500 bp DNA marker, lanes a1-a4 : human blood DNA (template DNA used to amplify the target genes GAPDH, TH01, TPOX and D7S820), with clear bands. Lanes b1-b4: mouse liver DNA (template used to amplify the target genes GAPDH, TH01, TPOX and D7S820), with no bands.