Although advances in microfluidic technology have enabled increasingly sophisticated biosensing and bioassay operations to be performed at the microscale, many of these applications employ such small amounts of charged biomolecules (DNA, proteins, peptides) that they must first be pre-concentrated to a detectable level. Efficient strategies for precisely handling minute quantities of biomolecules in microchannel geometries are critically needed, however it has proven challenging to achieve simultaneous concentration, focusing, and metering capabilities with currentgeneration sample injection technology. Using microfluidic chips incorporating arrays of individually addressable microfabricated electrodes, we demonstrate that DNA can be sequentially concentrated, focused into a narrow zone, metered, and injected into an analysis channel. The technique used in this research transports charged biomolecules between active electrodes upon application of a small potential difference (1 V), and is capable of achieving orders of magnitude concentration increases within a small device footprint. The collected samples are highly focused, with sample zone size and shape defined solely by electrode geometry. In addition to achieving the objectives of the research project, this setup was found to provide added functionality as a label-free biomolecule detection technique due to the formation of light scattering phases of charged biomolecules on top of the capture electrode.

published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

Ultraviolet radiation causes an alteration in DNA by modifying neighboring thymine bases resulting in the formation of a dimer. These dimers block the processes of transcription and translation and ultimately no protein is synthesized and the cell dies. However, DNA repair mechanisms correct this damage by excising the dimer from the DNA strand and inserting replacement bases which are joined to the original strand by DNA ligase. This allows transcription to resume and ultimately protein synthesis to take place.This research focused on determining the DNA damage and subsequent repair levels in a connective tissue disorder, namely Marfan syndrome. This information is important in understanding the clinical expression and management of life threatening conditions in Marfan syndrome individuals.Preliminary results indicate that at 20-25J/m2 UV dose (254nm) Marfan syndrome skin cells show a mean reduced survival value of 12% compared to normal human skin cells. Gel electrophoresis indicates a reduced DNA repair level 24h post UV irradiation for Marfan syndrome skin cells compared to normal human skin cells. These results suggest Marfan syndrome skin cells have reduced survival and DNA repair levels compared to normal human skin cells. / Department of Biology

Thesis (Ph. D.)--Ohio University, August, 2004. / Includes bibliographical references (p. 185-196).

Thesis (Ph.D.)--Ohio University, August, 2004. / Title from PDF t.p. Includes bibliographical references (p. 185-196)

A human liver cDNA library was screened by colony hybridization with two mixtures of synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of β-factor Xlla as predicted from the amino acid sequence. Four positive clones were isolated that contained DNA coding for most of factor XII mRNA. A second human liver cDNA library was screened by colony hybridization with ³²P-labeled cDNA clones obtained from the first screen and two identical clones were isolated. DNA sequence analysis of these overlapping clones showed that they contained DNA coding for the signal peptide sequence, the complete amino acid sequence of plasma factor XII, a TGA stop codon, a 3' untranslated region of 150 nucleotides, and a poly A⁺ tail. The cDNA sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the predicted amino acid sequence of factor XII, were identified three peptide bonds that are cleaved by kallikrein during the formation of β-factor Xlla. Comparison of the structure of factor XII with other proteins revealed extensive sequence identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like region and the kringle region) and fibronectin (type I and type II homologies). As the type II region of fibronectin contains a collagen-binding site, the homologous region in factor XII may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of factor XII shares considerable amino acid sequence homology with other serine proteases including trypsin and many clotting factors. A human genomic phage library was screened by using a human factor XII cDNA as ahybridization probe. Two overlapping phage clones were isolated which contain the entire human factor XII gene. DNA sequence and restriction enzyme analysis of the clones indicate that the gene is approximately 12 kbp in size and is comprised of 13 introns and 14 exons. Exons 3 through 14 are contained in a genomic region of only 4.2 kbp with introns ranging in size from 80 to 554 bp. The multiple regions found in the coding sequence of FXII that are homologous to putative domains in fibronectin and tissue-type plasminogen activator are contained on separate exons in the factor XII gene. The intron/exon gene organization is similar to the serine protease gene family of plasminogen activators and not to the clotting factor family. Analysis of the 5' flanking region of the gene shows that it does not contain the typical TATA and CAAT sequences found in other genes. This is consistent with the finding that transcription of the gene is initiated at multiple start sites. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Pichia pastoris is a methylotrophic yeast that is commonly used for its ability to express and secrete heterologous proteins. However, some proteins are not readily secreted in P pastoris and so adjustments in the secretion pathway must be made in order to achieve secretion. The Lin-Cereghino lab previously developed mutant strains using restriction enzyme-mediated integration that enabled P pastoris to secrete Pgalactosidase at higher levels than the wild type strain. This study focuses on characterizing the random pREMI-Z mutations in the genomic DNA and examining their secretory phenotype, in hopes of creating a super secretor strain. The ah3 mutant was specifically chosen and characterized for its ability to secrete HRP and SLPI proteins and the effect of the pREMI-Z mutation on the morphology of the cells using transmission electron microscopy. An examination into the AH3 protein yielded a comparative B-galactosidase secretion study between the ah3 mutant and ah3 mutant cells transformed with the pKANB-AH3 rescue construct. Lastly, a cell localization experiment was done to examine where the AH3 protein may be found. These experiments help to increase the current understanding of the secretion pathway in Pichia pastoris and serves as an outline of how to characterize other pREMI -Z mutant strains.

No description available.

The sexual assault kit backlog in the United States has become an increasing problem over the years. Combined with the number of kits laboratories receive with how it takes to extract the deoxyribonucleic acid (DNA) from the cells, it is hard for labs to keep up with the demand. The extraction method used is called differential extraction, where the epithelial cells from the victim are separated from the sperm cells from the perpetrator into different fractions. The Temperature Controlled Differential Extraction (TCDE) method is a novel procedure developed by the Cotton Lab at the Boston University School of Medicine and designed to decrease the extraction time while performing just as well, if not better, than traditional differential extraction methods. The TCDE method uses a series of temperature-controlled enzymes to lyse cells and purify the DNA extract. The purpose of this study is to compare this TCDE method to a method implemented by QIAGEN using the EZ1® Advanced biorobot for purification, which is used in many forensic laboratories. Ten female donors each received ten cotton swabs for vaginal cell collection; cotton swabs are typically found in sexual assault kits. Each swab then received either 5ng, 25ng, or 50ng of male DNA in the form of sperm cells. One half of the swab was processed using the TCDE procedure while the other half was processed using the EZ1® method. The TCDE method results in three fractions: the Epithelial Fraction (EF), the Material Fraction (MF), and the Sperm Fraction (SF). The EZ1® protocol was modified to include the additional MF. Results of both the quantitation data as well as the electropherograms (EPGs) produced are compared between the two methods. The quantitation data for the EF shows a variable amount of female DNA recovered due to the uncontrolled amount of female epithelial cells added to the swabs from the donors. The MF shows that large amounts of female epithelial DNA remain in the fraction for the EZ1® protocol and not the TCDE protocol because of the nuclease activity of one of the enzymes. The remaining male DNA on the MF can be used to compare to a known male profile, showing that there is valuable data potentially left behind. Regarding the SF, the EZ1® protocol resulted in a higher yield of DNA than the TCDE, however, the TCDE SF electropherograms are still able to be used for comparisons against known male profiles. The TCDE protocol cuts extraction time by almost half, and the quantitation results and EPGs prove that this method has the potential to become the new standard method of differential extraction.

Short Tandem Repeat (STR) analysis has become a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. This report describes the use of laser microdissection (LMD) for the separation of pure populations of spermatozoa from two-donor cell mixtures. In this study, cell separation was demonstrated by microscopic identification of histologically stained spermatozoa and female buccal cell mixtures, and STR analysis of DNA obtained from the separated sperm cells. Clear profiles of the male donor were obtained with the absence of any additional alleles from the female donor. Five histological stains were evaluated for use with LMD and DNA analysis: hematoxylin/eosin, nuclear fast red/picroindigocarmine, methyl green, Wright's stain, and acridine orange. Hematoxylin/eosin out-performed all other stains however nuclear fast red/picroindigocarmine could be used satisfactorily with STR analysis. In addition, three DNA isolation methods were evaluated for LMD collected cells: QIAamp (Qiagen), microLYSIS (Microzone Ltd.) and Lyse-N-Go (Pierce Chemical Co.). MicroLYSIS performed poorly, yielding low levels of PCR product. Lyse-N-Go extraction was effective for the recovery of DNA from LMD collected sperm cells while QIAamp isolation performed best for the recovery of DNA from LMD collected epithelial cells. LMD is shown to be an effective, low-manipulation separation method that enables the recovery of sperm while excluding epithelial cell DNA.