The multiplex PCR experiment is to amplify multiple regions of a DNA sequence at the same time by using different primer pairs. Although, in recent years, there are lots of methods for PCR primer design, only a few of them focus on the multiplex PCR primer design. The multiplex PCR primer design is a tedious task since there are too many constraints to be satisfied. A new method for multiplex PCR primer design strategy using genetic algorithm is proposed. The proposed algorithm is able to find a set of suitable primer pairs more efficient and uses a MAP model to speed up the examination of the specificity constraint. The dry-dock experiment shows that the proposed algorithm finds several sets of primer pairs for multiplex PCR that not only obey the design properties, but also have specificity.

Microalgae are photosynthetic organisms considered to be one of the most promising high-value chemicals and biofuel-producing organisms. However, there are several challenges for the widespread implementation of industrial processes using microalgae. The work presented in this dissertation proposes solutions to the different challenges involving the use of microalgae as microbial cell factories. To investigate the application of anaerobic digestion as a way to generate nutrients for microbial growth, salmon offal was used as substrate for anaerobic digestion, and soil from a flooded run-off pond on the Virginia Tech campus in Blacksburg, VA. A fast reduction in volatile solids and the short-chain fatty acid production profile is favorable for the growth of microalgae. A novel algae strain Scenedesmus sp. A6 was isolated from a decorative waterfountain in a hotel in Madison, IN. Mixotrophic growth trials were conducted using wastewater from the salmon offal digestion, that demostrated the A6 isolate grows six times faster in the wastewater then autotrophically. Bioassays of ethanolic cell extracts of A6 cultures demonstrated antimicrobial activity against E. coli cells at concentrations above 50 µg/ml. Genome sequencing and assembly revealed multiple copies of genes involved with acetate and ammonia metabolism, and several genes involved with secondary metabolite synthesis. An alternative to the high capital investment of photobioreactors for the cultivation of microalgae is the use of open-source and open-hardware bioreactor controller. Here, the concept of an open-hardwate bioreactor control called ``BioBrain'' is introduced. The BioBrain device is based on the Arduino Mega micro-controller board, and is capable of monitoring and controlling culture conditions during simple strain characterization studies, with an estimated construction cost of less than $800 USD. Finally, a new primer design tool for the ligation-independant cloning technique 𝜆-PCR was developed called lambdaPrimeR. The contributions of this work are the discovery and development of different tools that can overcome the challenges of the use of microalgae as microbial cell factories in industrial processes. / Ph. D. / Microalgae are single-celled organisms capable of photosynthesis and have the potential to revolutionize fuel and high-value chemical production. However, the high process costs involving the cultivation and biomass harvesting of these organisms limits the number of industrial applications of microalgae. Therefore, reduction of the overall costs of any process involving microalgae is vital for the widespread use of these organisms in industry. On this dissertation, I explore different approaches to tackle the challenges of using microalgae as a high-value chemicals cell factories. First, the use of anaerobic digestion of salmon offal to generate low-cost nutrients for algae growth is successfully demonstrated, with the discovery of a novel algae isolate Scenedesmus sp. A6, capable of very robust growth on the anaerobic digestion wastewater. Further characterization of this novel isolate showed that it has antimicrobial activity against E. coli cells. Therefore, the Scenedesmus sp. A6 isolate has the potential to be used as a high-value chemical cell factory. Reduction in equipment and instrumentation costs was also achieved by the design and construction of an open-hardware and open-source bioreactor controller device called the “BioBrain”, and a low-cost modular bubble column photobioreactor called “The Big Large Tube”. Together, these two devices represent a significant reduction in equipment costs for the cultivation of microalgae. Finally, an open-source Bioinformatics tool called “lambdaPrimeR” was developed to facilitate the use of a novel Genetic Engineering technique called λ-PCR, that has the potential to make genetic engineering of microalgae much easier.

<p>The desert ant Cataglyphis fortis lives in the Sahara desert where it is exposed to extreme temperatures up to 70° C. In other words, the organism is considered as a thermophile. Until now the genome remains unknown but the fact that C. fortis provides heat stable proteins makes it very interesting in the field of protein studies and maybe even therapeutical research later on. This thesis focuses on trying to find genes that are expressed in C. fortis. Different genes were chosen and capable primers designed. After fishing for the enzyme GAPDH a fragment was found and sequenced. The sequence showed 31% homology on amino acid level with protein disulfide isomerase (PDI) in Apis mellifera (honey bee) and Drosophila melanogaster (fruitfly). The received sequence can be used to design new primers that match exactly. Gene fishing can also be continued by using the other primers that were designed during this project.</p>

<p>Sex chromosomes are useful in elucidating the evolutionary factors affecting diversity and divergence. In particular, Y chromosome analyses may complement studies using mitochondrial DNA for inferring sex-specific population genetic processes.</p><p>Y chromosome studies have been scarce due to limited access to genetic markers and the dynamic evolution of Y. Conserved Y-specific primers that could amplify a diverse set of mammalian species were developed from comparison of gametologous X and Y sequences. Y-specific sequence, generally more than one kb, was amplified for all 20 species examined.</p><p>Intraspecific diversity on mammalian Y was found to be reduced even when male-biased mutation rate and effective population size were corrected for. A number of factors can cause this low variation on Y of which selection on a haploid chromosome seems most important.</p><p>The field vole (<i>Microtus agrestis</i>), a common and well-studied small mammal in Eurasia, was examined for X and Y variability. Earlier studies on mtDNA had shown that the field vole is separated in two distinct lineages in Europe. The X and Y chromosome sequences confirmed the deep split and suggested that the two lineages of field vole should be reclassified as two separate species.</p><p>Two distinct Y chromosome haplogroups were found in modern European cattle, distributed among breeds according to a north-south gradient. Ancient DNA analysis of European aurochsen showed the northern haplogroup to be the most common, possibly indicating local hybridization between domestic cows and wild aurochs bulls in Europe.</p>

Sex chromosomes are useful in elucidating the evolutionary factors affecting diversity and divergence. In particular, Y chromosome analyses may complement studies using mitochondrial DNA for inferring sex-specific population genetic processes. Y chromosome studies have been scarce due to limited access to genetic markers and the dynamic evolution of Y. Conserved Y-specific primers that could amplify a diverse set of mammalian species were developed from comparison of gametologous X and Y sequences. Y-specific sequence, generally more than one kb, was amplified for all 20 species examined. Intraspecific diversity on mammalian Y was found to be reduced even when male-biased mutation rate and effective population size were corrected for. A number of factors can cause this low variation on Y of which selection on a haploid chromosome seems most important. The field vole (Microtus agrestis), a common and well-studied small mammal in Eurasia, was examined for X and Y variability. Earlier studies on mtDNA had shown that the field vole is separated in two distinct lineages in Europe. The X and Y chromosome sequences confirmed the deep split and suggested that the two lineages of field vole should be reclassified as two separate species. Two distinct Y chromosome haplogroups were found in modern European cattle, distributed among breeds according to a north-south gradient. Ancient DNA analysis of European aurochsen showed the northern haplogroup to be the most common, possibly indicating local hybridization between domestic cows and wild aurochs bulls in Europe.

The human genome is predicted to contain ~20,500 protein-coding genes. The encoded proteins are the key players in the body, but the functions and localizations of most proteins are still unknown. Antibody-based proteomics has great potential for exploration of the protein complement of the human genome, but there are antibodies only to a very limited set of proteins. The Human Proteome Resource (HPR) project was launched in August 2003, with the aim to generate high-quality specific antibodies towards the human proteome, and to use these antibodies for large-scale protein profiling in human tissues and cells. The goal of the work presented in this thesis was to evaluate if antigens can be selected, in a high-throughput manner, to enable generation of specific antibodies towards one protein from every human gene. A computationally intensive analysis of potential epitopes in the human proteome was performed and showed that it should be possible to find unique epitopes for most human proteins. The result from this analysis was implemented in a new web-based visualization tool for antigen selection. Predicted protein features important for antigen selection, such as transmembrane regions and signal peptides, are also displayed in the tool. The antigens used in HPR are named protein epitope signature tags (PrESTs). A genome-wide analysis combining different protein features revealed that it should be possible to select unique, 50 amino acids long PrESTs for ~80% of the human protein-coding genes. The PrESTs are transferred from the computer to the laboratory by design of PrEST-specific PCR primers. A study of the success rate in PCR cloning of the selected fragments demonstrated the importance of controlled GC-content in the primers for specific amplification. The PrEST protein is produced in bacteria and used for immunization and subsequent affinity purification of the resulting sera to generate mono-specific antibodies. The antibodies are tested for specificity and approved antibodies are used for tissue profiling in normal and cancer tissues. A large-scale analysis of the success rates for different PrESTs in the experimental pipeline of the HPR project showed that the total success rate from PrEST selection to an approved antibody is 31%, and that this rate is dependent on PrEST length. A second PrEST on a target protein is somewhat less likely to succeed in the HPR pipeline if the first PrEST is unsuccessful, but the analysis shows that it is valuable to select several PrESTs for each protein, to enable generation of at least two antibodies, which can be used to validate each other. / QC 20100705

The desert ant Cataglyphis fortis lives in the Sahara desert where it is exposed to extreme temperatures up to 70° C. In other words, the organism is considered as a thermophile. Until now the genome remains unknown but the fact that C. fortis provides heat stable proteins makes it very interesting in the field of protein studies and maybe even therapeutical research later on. This thesis focuses on trying to find genes that are expressed in C. fortis. Different genes were chosen and capable primers designed. After fishing for the enzyme GAPDH a fragment was found and sequenced. The sequence showed 31% homology on amino acid level with protein disulfide isomerase (PDI) in Apis mellifera (honey bee) and Drosophila melanogaster (fruitfly). The received sequence can be used to design new primers that match exactly. Gene fishing can also be continued by using the other primers that were designed during this project.

Exons are expression DNA sequences. A DNA sequence which includes gene has exons and introns. During transcription and translation, introns will be removed, and exons will remain to become protein. Many researchers need exon primers for PCR experiments. However, it is a difficult to find that many exon primers satisfy all primer design constraints at the same time. Here, we proposed an efficient exon primer design algorithm. The algorithm applies multiobjective genetic algorithm (MGA) instead of the single objective algorithm which can easily lend to unsuitable solutions. And a hash-index algorithm is applied to make specificity checking in a reasonable time. The algorithm has tested by a variety of mRNA sequences. These dry dock experiments show that our proposed algorithm can find primers which satisfy all exon primer design constraints.

Made available in DSpace on 2014-12-17T14:56:06Z (GMT). No. of bitstreams: 1 KligerKFR.pdf: 1442515 bytes, checksum: b8c82b51681c5740727addb5f0eed20a (MD5) Previous issue date: 2005-04-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / It proposes a established computational solution in the development of a software to construct species-specific primers, used to improve the diagnosis of virus of plant for PCR. Primers are indispensable to PCR reaction, besides providing the specificity of the diagnosis. Primer is a synthetic, short, single stranded piece of DNA, used as a starter in PCR technique. It flanks the sequence desired to amplify. Species-specific primers indicate the well known region of beginning and ending where the polymerase enzyme is going to amplify on a certain species, i.e. it is specific for only a species. Thus, the main objective of this work is to automatize the process of choice of primers, optimizing the specificity of chosen primers by the traditional method / Prop?e-se uma solu??o computacional baseada no desenvolvimento de um software para construir primers esp?cie-espec?ficos, usados para melhorar o diagn?stico de viroses de planta por PCR. Primers s?o indispens?veis ? rea??o PCR, al?m de proporcionar a especificidade do diagn?stico. Um primer ? um fragmento de DNA sint?tico, curto e de fita simples, utilizado como um iniciador na t?cnica PCR que flanqueia a seq??ncia que se deseja amplificar. Primers esp?cie-espec?ficos s?o primers que s? indicam a regi?o bem conhecida de in?cio e t?rmino onde a enzima polimerase vai amplificar, de uma determinada esp?cie, ou seja, ? espec?fica para somente uma esp?cie. Assim, o objetivo principal deste trabalho ? automatizar o processo de escolha de primers, otimizando a especificidade dos primers escolhidos pelo m?todo tradicional

<p>Loop-mediated Isothermal Amplification (LAMP) is a promising technology to address diagnostic and surveillance testing during public health crises, such as the current COVID-19 pandemic; however, primer design and assay optimization remain a barrier to enabling rapid deployment of assays based on LAMP. Herein, we introduce a design and screening process that allows for strategic determination of optimally performing primer sets and standardized assay conditions which enable execution of LAMP at point-of-care (POC) settings using complex human samples such as saliva. A total of 20 primer sets targeting the N, E, RdRP, and orf1ab genes of the SARS-CoV-2 were designed, screened, and selected based on performance metrics such as reaction time, sensitivity, and specificity. Of these 20 primer sets, only two primer sets (orf1ab.2 and orf1ab.4) proved to be viable for use in the final assay. Colorimetric RT-LAMP of the selected primer set, orf1ab.2 was shown to produce a distinct color change in contrived samples containing heat-inactivated SARS-CoV-2 in 5% saliva. The limit of detection of our assay using primer set orf1ab.2 was determined to be 1000 copies/µL of saliva collected. Furthermore, methods are introduced which allow for the high-throughput design of LAMP primers using standard software tools and the <i>in-silico</i> performance of LAMP primer sets. </p>