These levels would have been helpful in determining if the primary study results were due to increased nicotine exposure in smokers with heavy caffeine or marijuana use. And third, some smokers had small measurable plasma nicotine levels at the time of scanning , which led to mathematical corrections for these levels. While overall study results did not differ with or without these corrections, an improved method of ensuring nicotine abstinence could have been helpful. Additionally, in the exploratory analysis from our previous study , lower caffeine use was associated with greater nAChR availability. Results from this prior exploratory analysis of a group with modest caffeine use would not have passed Bonferroni correction. In contrast, the finding here of greater nAChR availability in heavy caffeine users was highly significant . Thus, the present findings indicate a robust elevation of nAChR availability in heavy caffeine using smokers. In conclusion, smokers with concomitant heavy caffeine or marijuana use have greater α4β2* nAChR availability than smokers without such heavy use. These findings are consistent with prior research demonstrating more severe dependence on cigarettes in caffeine and marijuana users .DNA analysis can identify the biological source of archaeological artifacts. This is true for many plant-based artifacts. Plant cells contain plastids, such as chloroplasts in leaves-often many copies and plastids contain DNA sequences that are useful for identification. There is a great deal of information available concerning the base sequences of plastid genes in different plants, much of it gathered for use in determining evolutionary relationships. This information can be applied to objects like textiles and baskets. At first glance,hydroponics system for cannabis it may be surprising that DNA persists in manufactured objects, and some processes-e.g, mordanting-do break down DNA. However, even present-day rope is made with natural fibers that receive a minimum of treatment, and the rope contains fragments of tissue with intact organelles .
We expect that treatments of fibers in the past were less stringent and the products from which they were made more likely to retain plastids and nuclei. It may be even more surprising that DNA persists in ancient objects, since we can expect the rigors of time, with accompanying hydration, desiccation, and temperature extremes, to break down biological molecules. In fact, that does occur . But DNA may show a degree of resistance under certain conditions. Indeed its structure may have evolved in part to increase its stability . There have been many reports of ancient DNA isolated from, for example, mammoths preserved in glaciers , human mummies , wood , and rope . The degradation of DNA results in a gradual reduction in its length as the polymeric strands become fragmented, but even relatively short fragments retain useful information in their base sequences. With those considerations in mind, we resolved to identify DNA in fibers of samples of rope and cloth that have been found in an archeological site near Qumran and the Dead Sea. Microscopic observations have identified various samples of cloth from caves above Qumran as flax, cotton, and wool . Muller et al. , using X-ray micro-diffraction, identified flax and cotton in Qumran samples. But there have been indications of the early use of hemp in East Asia and in Europe and Asia Minor . Our objective was to learn whether the DNA of materials from an archeological site near the Dead Sea could confirm the presence of hemp or other fibers. We expected the use of DNA sequence information to confirm the identity of the major component , but also to indicate whether fibers from hemp or another plant species form a detectable fraction of one or more samples. As will be shown, our data do indicate that flax-linen dominates in every sample tested and that there is a small amount of hemp DNA in most samples.As noted in the Methods section, the choice of primers was a critical part of the study. Following the discovery that no rbcL DNA product 771 base pairs long could be amplified from the archeological DNA templates, we tested various rbcL primer combinations to find a successful set . Using template DNA from modern hemp, amplified DNA was obtained with all four primer pairs tested. However, templates from flax plants and from modern flax rope only gave product using primer pairs rbcLF2/rbcLR3a and rbcLF2/rbcLR4a. Three template DNAs extracted from archeological samples gave product corresponding to the smallest band obtained with flax.For subsequent tests, we concentrated on rbcLF2/rbcLR3a, which gave the smallest product and thus was least likely to discriminate against the gene from a minor species. The rbcL PCR products obtained using the template DNA extracted from all the archeological cordage and textile samples contained strong bands of approximately 184 base pairs .
To distinguish between flax and hemp templates, we noted that the band produced using authentic C. sativa DNA template was cut over 90% by BamH1, yielding fragments of 115 and 69 base pairs. The PCR product of the modern UK rope did not show a detectable amount of cutting and thus was entirely flax. Interestingly, the PCR product of the modern Japanese rope showed a small amount of cutting, indicating the presence of some C. sativa DNA. Most of the archeological samples showed only faint bands at 115 and 69 base pairs after BamH1 treatment, indicating that, like the Japanese rope, they contained little C. sativa DNA. Rope sample 931 and textile sample 786 were the most notable exceptions, with sample 931 showing 44% cutting and sample 786 showing 39% cutting. However, repetitions of the PCR reaction and restriction digestion, particularly of sample 786, did not consistently show the smaller bands produced by BamH1. The base sequences of DNA from the archeological samples confirmed their identity as primarily L. usitatissimum Fig. 6. Within the 184-base pair amplified DNA, there was a stretch of 86 base pairs in which accurate sequence determinations could be obtained from both primers. Within that region were nine sites at which the sequences of the Linum and Cannabis genes differed. These quence of rope sample 931 showed super positions of two bases at all nine sites , confirming that this sample contained a significant amount of Cannabis DNA. Chromatograms of the other samples, including textile sample 786, were not interpreted by the computer as having a significant amount of Cannabis DNA, but small peaks corresponding to Cannabis bases could be seen in the chromatogram for sample 786 . A few other base-sequence super positions, e.g. K or a present/deleted base , occurred near the ends of the 86- base pair stretch, but probably represented sequencing errors rather than the inclusion of a variant Linum or another species, since in each case the super positions were found with only one primer. However, one of the super positions in sample 931 indicated C/T , whereas the sequences of Linum and Cannabis at that position were C and A,respectively. In this position, the codons containing C, T, and A all code for glycine, so this is a “silent” substitution. It is possible that the rbcL gene of the archeological Cannabis differed from the modern species used for identification. In an attempt to confirm the data obtained from the rbcL analysis, we used the extracts of DNA as templates to amplify fragments of two other chloroplast genes, trnL and matK. Using the trnL primers, we obtained Cannabis fragments from rope sample 931 and most of the textile samples, especially 786 .
There was no indication of Linum template DNA in the ancient samples, although the modern control gave a good band. Using the matK primers, we could not obtain Linum or Cannabis fragments from any archeological DNA templates, although again the modern control templates worked well . Assuming that the primer-complementary sequences of the trnL and matK target genes have not changed drastically over the last 2000 years, these results suggest that different regions of chloroplast DNA fragment at different rates. Finally, we estimated the relative amounts of flax and hemp DNA using a semi-quantitative “kinetics” technique in which we compared the amount of rbcL PCR product as a function of the number of replication cycles. Although under appropriate conditions,indoor hydroponics cannabis the amplification of DNAs can give amounts of products that represent the relative amounts of different templates, many problems with the technique can confound the analysis. It is more accurate to compare the number of cycles that give equal band intensities upon staining, even given the uncertainty inherent in the relationship between staining intensity and DNA size. We applied this technique to three samples that appeared to show three different levels of hemp DNA, 931, 019, and 928. In fact, as shown in Fig. 9, the levels of hemp varied from ca one-fourth that of flax to 1/4000 .The PCR data confirmed the identities of the contents of all the cordage and textile samples as primarily flax-linen. The sequence data revealed the presence of hemp DNA, and by inference hemp fibers, but only in one rope sample, 931, and one textile sample, 786. The gel electrophoresis patterns, more sensitive to minor components, indicated the presence of hemp DNA in those samples and all the other samples, with the exception of the control Linum DNA and the DNA from the contemporary flax rope. The lack of linearity inherent in PCR made it impossible to estimate the relative amounts of flax and hemp accurately from the initial analyses, which were performed with a fixed number of amplification cycles, but a modification of the PCR protocol revealed that the fraction of DNA from hemp varied widely, from 25% to 0.025% that of the amount of flax DNA. The ubiquity of the hemp DNA, particularly in the small amounts found in most of the archeological samples, forces us to consider the possibilities for its origin. These include deliberate incorporation of hemp into flax rope and linen textiles in situ and the importation of hemp containing rope and textiles from other places. Samples 931 and 786 most likely acquired their substantial amounts of hemp DNA in their fabrication. Samples that had much smaller amounts of hemp DNA might have acquired it as dust, through their storage over several centuries in contact with some of hemp products. In the same way, samples might have acquired small amounts of hemp dust during their excavation and transport to museums, particularly if their bundles were bound with hemp rope or twine.
Finally, we cannot ignore the possibility of contamination in the analytical laboratory, although controls did not indicate this. The lack of contemporary hemp bands in the archeological samples in Fig. 4 strongly suggests that the hemp DNA, whether incorporated deliberately or through contamination, was ancient. What is the possibility that the results we interpret as representing hemp actually reflect another fiber? A comparison of the amplified segments of the rbcL genes from six old-world fiber plants, flax, hemp, date palm, cotton, banana palm, and ramie, shows that the base sequences of the central regions differ among all six. The BamH1 site in the hemp sequence occurs only in that species, so that the minor bands seen in Fig. 5 must represent hemp. The specific base super positions shown in Fig. 7 occur only in the flax-hemp pairing. The lack of other minor bases indicates that, if present, the amounts of other fibers must be very small. The 14C dating of the samples confirms a suggestion by Dr. Orit Shamir that they represent two distinct periods of use, one prehistoric and one from the time of the Roman conquest. Previous 14C dating of two wood samples from CC found even earlier dates, 3670 and 4830 BCE , but the authors pointed out that the dates might have represented “cultural activity 6000 years ago or the use of old wood.” Samples from the “Cave of the Warrior,” dated to approximately the same times, ca. 3800 BCE and ca. 4400 BCE , included textiles and baskets, a clear indication of prehistoric culture. Our three dated samples from the Early Bronze period, though somewhat younger, confirm the suggestion of very old cultural activity and add the information that this activity included the use of hemp. In conclusion, this work points out the value of PCR in determining the plant-fiber composition of textile and cordage, particularly when there are mixtures of fibers.