Browsing by Subject "Plant Breeding and Genetics"
Now showing 1 - 3 of 3
- Results Per Page
- Sort Options
Item Amplified Fragment Length Polymorphism Analysis of White Oak Tree Leaves(2005-07-01) Patel, Kaajal Devendra; John Planz; Joseph Warren; Arthur EisenbergThe AFLP technique at first seems to be a remarkable new technology that can be applied to the growing area of non-human DNA testing. The ability to identify organisms without prior genetic knowledge would be an asset to a field such as non-human DNA testing since not enough research in the area is being conducted. With any new technique or theory in science, intense scrutiny must be used to examine the applicability of the new technology. In the area of forensic science, the severe consequences of a false result extend far beyond the realm of scientific error. Errors make in forensic casework could result in life changing occurrences for the families of not only the victim, but the defendant as well. From this study it can be seen that AFLP as a technique may not stand up to the high expectations of reliability, and reproducibility required for a technique to be adopted into the field of forensic science. Several problems occurred through this study that may prevent this technology from becoming a widely accepted technique in non-human DNA testing. The initial problems with the technique were associated with reproducible results. The first several attempts were conducted under the same conditions, by the same analyst but yielded results that were no comparable. The RFUs of each experiment were inconsistent, not only between samples examined at different times, but samples examined within the same tiral as well. AFLP as a technique is supposedly insensitive to template concentrations however, it has been previously shown to produce differences in the electropherogram when the template is excessively diluted (26). Vos et al. (1995) determined that high dilutions yielding template DNA concentrations below 1 pg could result in irreproducible fingerprints. In this study 27.5 ng of template DNA was added to each digestion-ligation reaction, yet the resulting quantity of amplified fragments varied. These variations in quantities of amplified product could be due to PCR inefficiencies when comparing samples from different trials, but it does not explain instances where duplicate trials were inconsistent with each other (10, 22). When new ligase was introduced the resulting electropherograms did produce considerably higher RFUs for each peak, but the lack of interpretable peaks observed previously may not have been solely due to inefficient ligase. In an inter-laboratory study, Jones et al. (1997) noted that several laboratories encountered problems in obtaining complete AFLP profiles. For several groups, up to 50% of the bands were missing during the preliminary testing. Though this problem subsided with successive attempts, this approach to achieving successful results may not be feasible in a forensic setting. Often the evidence received from a crime scene may be insufficient to allow for multiple testing. In addition, multiple attempts to obtain results may open up areas for scrutiny and attack by the defense counsel. Repetitive testing may appear to be a biased search for condemning evidence against the questioned party, rather than the production of reliable results. Repetitive testing may also not be possible since laboratory reagents and time involved in the production of these results may not be within the constraints of a crime laboratory. In this study, capillary electrophoresis was used to visualize the fluorescent dyes attached to each fragment however, laboratories could use radioisotopes and polyacrylamide gels instead. This method of visualizing AFLP fingerprints is not only costly, but time consuming as well. Conducting repetitive tests in order to obtain a sample with sufficiently intense bands for analysis may not be feasible. These limitations may therefore restricts the use of the AFLP technique from only being conducted in laboratories with sufficient time and funds to conduct repetitive testing as is needed (10). Despite the potential cost in time and funds, the technique was able to produce AFLP fingerprints that were consistent with each other when the electropherograms were compared. The major source of error resulted from the method used to determine the presence of peaks within the designated categories. Since not all peaks crossed the 50 RFU detection threshold, they were not identified by the Genotyper macros. However, when the actual electropherograms were compared, these peaks were present. It has been suggested that to verify whether each peak is present in the pre-designated categories a scan of the electropherogram should be done and any peaks that were not called by the macro should be manually entered into the binary table or should be reanalyzed (Heather Coyle, personal communications). Although this method could potentially aid in the correct genotyping of each sample, it requires a considerable amount of user intervention. A considerable amount of time is needed to examine each electropherogram for the presence of peaks that are below the 50 RFU threshold. Without a redefined interpretation threshold, the analysis of each electropherogram can be highly subjective. Peaks that are relatively low need to be distinguished from peaks that may be associated with background noise. Therefore, in order to eliminate analyst bias a peak detection threshold must be established. Generally the interpretation threshold is established by a validation study of the analysis technique. In this study the lower threshold was previously established at 50 RFU for the instrument being used, but this threshold was insufficient for the recognition of all peaks present during the AFLP analysis. The question then becomes to what extend the peaks can or should be called in order to correctly identify each organism without errors. The exclusion of some peaks could lead to discrepancies, such as those observed during the blind study, which could result in an initial false match or exclusion. The interlaboratory study by Jones et al. found only one scoring difference associated with the absence of one band out of a total of 172 in the AFLP profiles. This error was later associated with experimental errors that incurred during the AFLP procedure. Discrepancies such as this can lead to an erroneous identification of samples that could have severe consequences in a criminal case. At this time, the utilization of AFLP technique for further testing of other organisms such as Cannabis sativa does not seem feasible. A variety of adjustments in the technique need to be addressed before this technology should be further applied to organisms in forensic casework. In order for AFLP typing to be used for forensic casework, major improvements in the technique need to be made. Consistency in obtaining reliable electropherograms with peaks well above the RFU detection threshold must be resolved in order to allow for accurate sample interpretation. This will not only allow for greater consistency between replicates, but will also help in establishing new databases for organisms that are being tested. As with any type of forensic DNA analysis, a database must be established for each organism being tested. Without a reliable database, accurate identification of crime scene evidence cannot be established. A major improvement that is required for the utilization of AFLP typing is the process by which genotypes are identified. Utilizing the macros to identify control and variable peaks to create the binary table was a quick and easy method, however it was not always able to identify the correct genotype. The overlapping of electropherograms in GeneScan ultimately was the best method for accurate identification of the blind samples, but in a real case scenario it would not be feasible to compare each evidentiary electropherogram with those in a database. Advancements in technology will continually introduce new techniques and procedures that could be applicable to the field of forensic science. As with any new technique, the methods and theories must be validated in order to determine whether they can be used in a criminal case. The field of non-human DNA testing is growing and with the advent of new technology such as AFLP, the possibility for establishing a non-human DNA identification method may be on the horizon.Item Genetic Engineering and the Food Supply(2000-12-01) Kennedy, W. Russ; Samuel F. AtkinsonAs food sources high in the trees became inadequate, our predecessors climbed to the ground and through centuries of adaptation learned to stand upright and cultivate plants. Plant cultivation has been practiced for more than ten thousand years with continuous improvements made to crop plants to meet the growing food needs of human domesticated animal populations. Biotechnology has been practiced for thousands of years with fermentations of fruits and grains to make wine and beer and the use of yeast in baking. More recently, advances in molecular biology allow the analysis and manipulation of genetic material to achieve desired changes in the organism. Transgenics or genetic engineering is the process of identifying specific genetic defects or desirable traits and altering an organism’s DNA by addition or deletion of specific DNA sequences. Nearly 100 million acres (40 million hectares) were planted in transgenic crops in 1999. The largest acreages of more than 40 different transgenic crops grown were in cotton, corn, soybean and rapeseed. Fifty-five percent of all cotton, 50% of soybeans, and 33% of corn grown in the U.S. in 1999 were transgenic varieties. The large plantings stem from fairly straightforward manipulations of single genes, such as the transferring to corn and cotton genetic material from the bacterium Bacillus thuringiensis (Bt) which produces an insecticidal toxin or transferring to the soybean, corn, cotton, sugar beets, and canola a gene with resistance to herbicides, such as glyphosate. The American farmer is perceived to be the beneficiary of lowered production costs primarily through better weed and pest control and a reduction in pesticide use with accompanying environmental improvement. Agro-chemical companies, who for the most part have spearheaded research and development of these crops, became involved because they foresaw a declining market for pesticides. Another area of promise widely discussed in the scientific and popular press is the improvement of food quality and composition resulting from genetic engineering. Because plants and plant products provide much of the world’s food supply, it is only fitting that early applications of this technology be in this area. Recent estimates suggest that the market for transgenic seed has already reached several hundred million dollars per year and that more than 15 million hectares (37 million acres) were grown in the U.S. in 1998. Concerns of food risk to the food supply and environment that using transgenic methods present, although not always science based, have some merit and require careful scientific scrutiny.Item Quorum Sensing in Sinorhizobium meliloti(2008-12-01) Patankar, Arati V.; Juan E. Gonzales; Jerry W. Simecka; Stephen O. MathewPatankar, Arati V., Quorum Sensing in Sinorhizobium meliloti. Doctor of Philosophy (Microbiology and Immunology), December 2008, 170 pp., 14 tables, 23 illustrations, bibliography, 212 titles. The overall goal of this study was to elucidate the role of a series of transcriptional regulators and potential signal molecules in the coordination of gene regulation in Sinorhizobium meliloti. The agriculturally important gram-negative soil bacterium S. meliloti, forms a symbiotic association with its host legume, Medicago sativa (alfalfa); thereby serving as a good model for studying host-bacterial interactions. Often, bacteria associated with eukaryotic hosts utilize global gene regulatory systems to coordinate their behavior in order to establish pathogenic or symbiotic associations. Quorum sensing is one such form of bacterial gene regulation which is mediated by signaling molecules and regulatory proteins in a population density dependent manner. In S. meliloti, the process of quorum sensing has been shown to play an important role in the relationship with its host plant. Control of essential processes such as plant nodulation and exopolysaccharide production has been attributed to the Sin/ExpR quorum-sensing system of S. meliloti. Interestingly, S. meliloti contains four additional (SMc04032, SMc00658, Smc00878 and SMc00877) putative quorum-sensing response regulators whose regulatory network was not known. The predicted protein sequences of these genes contain features typical of the LuxR family of proteins i.e., an N-terminal signal binding domain and C-terminal helix-turn-helix DNA biding domain. In order to identify their regulatory role, mutants of the response regulators were constructed and their expression profile was determined by employing genome-wide microarray and real-time PCR expression analysis. Through these analyses, it was determined that the SMc004032 locus controls expression of genes involved in the active methyl cycle, while the SMc00658, SMc00878 and SMc00877 loci control expression of genes from the denitrification of pathway of S. meliloti. Further, through phenotypic studies it was established that SMc04032 impacts stress response adaptation, and effective competition for plant nodulation. This suggests that SMc04032 could play a role in bacterial survival in the soil as well as within the host. The ability to denitrify is highly variable in different strains of S. meliloti. Through growth and enzymatic assays, it was established that the wild-type strain of this study, S. meliloti Rm8530, is a partial dentrifier in which, the capacity to metabolize nitrate is impaired. It was further determined that SMc00658, SMc00878 and SMc0877 modulated nitrite reductase activity under aerobic conditions, implying that these genes are involved in aerobic denitrification and therefor probably play a role in detoxification in S. meliloti. Based on the sequenced-genome analysis, S. meliloti possess homologs of other mediators of quorum sensing, that might be responsible for the synthesis of novel signal molecules. Bioreporter strains and mass spectrometry analysis were employed to identify production of cyclic dipeptides in S. meliloti. These compounds have been previously reported as quorum-sensing signal molecules in several bacteria. The results presented in this study provide a better understanding of S. meliloti’s metabolic and physiological properties and will be fundamental in future studies of bacterial interaction with its host and survival within its ecological niche.