Minimizing the surface effect of PDMS–glass microchip on polymerase chain reaction by dynamic polymer passivation

Polydimethyl siloxane (PDMS)–glass microchip has a very strong surface effect on polymerase chain reaction (PCR), leading to a very poor PCR yield. In the work reported here, practical dynamic passivation of surfaces of PDMS–glass microchip using polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP) was achieved using a conventional thermocycler. The passivation procedure was cost-effective and easy to conduct. The effects of polymer molecular weight and polymer concentration on tube PCR efficiency were investigated primarily to prescreen out suitable polymers and polymer concentrations in the PCR mixture. The result from tube PCR indicated that both PEG and PVP could affect the performance of Taq polymerase. A final concentration of 0.025% (w/v) or 0.4% (w/v) polymer in the PCR mixture can enhance the tube PCR, while 1% (w/v) polymer was found to inhibit the reaction. PEG was more effective in tube PCR, although PVP performed better in chip PCR. Instead of employing the polymer directly in the PCR mixture, i.e. the conventional in situ passivation approach, another approach of dynamic passivation by pre-injecting polymers into the microchip achieved better performance. The efficiency of pre-passivation was found to follow the order: PVP10000>PVP55000, PEG8000> PEG10000>PEG400. After pre-passivation with PVP10000, PVP55000 and PEG8000, the PCR efficiency can recover to 93%, 86% and 83%, respectively, of that obtained from tube PCR. Copyright © 2006 Society of Chemical IndustryPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55877/1/1631_ftp.pd

Mutations in VPS13D lead to a new recessive ataxia with spasticity and mitochondrial defects

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145306/1/ana25220_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145306/2/ana25220.pd

Genome-Wide Analysis of Central Corneal Thickness in Primary Open-Angle Glaucoma Cases in the NEIGHBOR and GLAUGEN Consortia

To investigate the effects of central corneal thickness (CCT)-associated variants on primary open-angle glaucoma (POAG) risk using single nucleotide polymorphisms (SNP) data from the Glaucoma Genes and Environment (GLAUGEN) and National Eye Institute (NEI) Glaucoma Human Genetics Collaboration (NEIGHBOR) consortia

Theoretical and Experimental Investigation of the Impact of Surfaces on DNA Melting Temperature.

The design of microarrays rely on studies geared towards sequence-specific recognition between complementary probe and target molecules in bulk solution. However, this proves to be insufficient to understand the duplex formation reaction on solid-phase. In this dissertation, influence of the surface on DNA duplex stability and melting temperature were theoretically and experimentally investigated. The theoretical approach represents electrostatic and entropic repulsions experienced by hybridizing targets. Electrostatic blocking stemming from surface charge was modeled through Electric Double Layer Theory and Surface Partition Model. Entropic blocking due to steric effects was modeled using polymer physics. Investigated experimental parameters were target concentration, spacer length and probe density. All the experiments gave reproducible melting temperatures with values lower on-surface than in-solution. In a representative set, a target concentration increase from 0.5nM to 15nM with 0.82pmoles of probe at 5*10^12 molecules/cm2 density on 15 dT spacer resulted in approximately 8°C decrease in melting temperature, compared to 5°C increase in solution. This decreasing trend was supported by theory with increasing steric and electrostatic effects at increasing target concentrations leading to higher hybridization efficiencies. Additionally, at low target concentrations (0.0165nM), we observed a multiple melting process in low temperature domains of melting curves due to low stability truncated probes; an indirect indication of synthesis quality. It was observed that as spacer length increases from 2 dT to 25 dT with 0.82pmoles of probe at 5*10^12 molecules/cm2 density with target concentrations ranging from 0.36nM to 1nM, melting temperature increases; an observation theoretically explained by possible entropic blocking dominance. Probe density effect was tested at 5*10^12 molecules/cm2 and 5*10^13 molecules/cm2, on 15 dT spacer and target-to-probe concentration ratios of 0.61:1 to 1.7:1. It was observed that high probe density resulted in lower melting temperature. This trend was theoretically supported by increasing electrostatic and crowding effects. Previously observed dependence of melting temperature on target concentration was also confirmed in all experiments. Melting temperature dependence on probe density seems to be stronger than the dependence on spacer length. The results of this work would lead to better experimental design and correct use of microarrays.Ph.D.Chemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/62212/1/aozel_1.pd

Target concentration dependence of DNA melting temperature on oligonucleotide microarrays

The design of microarrays is currently based on studies focusing on DNA hybridization reaction in bulk solution. However, the presence of a surface to which the probe strand is attached can make the solution‐based approximations invalid, resulting in sub‐optimum hybridization conditions. To determine the effect of surfaces on DNA duplex formation, the authors studied the dependence of DNA melting temperature (T m ) on target concentration. An automated system was developed to capture the melting profiles of a 25‐mer perfect‐match probe–target pair initially hybridized at 23°C. Target concentrations ranged from 0.0165 to 15 nM with different probe amounts (0.03–0.82 pmol on a surface area of 10 18 Å 2 ), a constant probe density (5 × 10 12 molecules/cm 2 ) and spacer length (15 dT). The authors found that T m for duplexes anchored to a surface is lower than in‐solution, and this difference increases with increasing target concentration. In a representative set, a target concentration increase from 0.5 to 15 nM with 0.82 pmol of probe on the surface resulted in a T m decrease of 6°C when compared with a 4°C increase in solution. At very low target concentrations, a multi‐melting process was observed in low temperature domains of the curves. This was attributed to the presence of truncated or mismatch probes. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/91159/1/1505_ftp.pd

Analyses of LMNA-negative juvenile progeroid cases confirms biallelic POLR3A mutations in Wiedemann-Rautenstrauch-like syndrome and expands the phenotypic spectrum of PYCR1 mutations

Juvenile segmental progeroid syndromes are rare, heterogeneous disorders characterized by signs of premature aging affecting more than one tissue or organ starting in childhood. Hutchinson-Gilford progeria syndrome (HGPS), caused by a recurrent de novo synonymous LMNA mutation resulting in aberrant splicing and generation of a mutant product called progerin, is a prototypical example of such disorders. Here, we performed a joint collaborative study using massively parallel sequencing and targeted Sanger sequencing, aimed at delineating the underlying genetic cause of 14 previously undiagnosed, clinically heterogeneous, non-LMNA-associated juvenile progeroid patients. The molecular diagnosis was achieved in 11 of 14 cases (similar to 79%). Furthermore, we firmly establish biallelic mutations in POLR3A as the genetic cause of a recognizable, neonatal, Wiedemann-Rautenstrauch-like progeroid syndrome. Thus, we suggest that POLR3A mutations are causal for a portion of under-diagnosed early-onset segmental progeroid syndromes. We additionally expand the clinical spectrum associated with PYCR1 mutations by showing that they can somewhat resemble HGPS in the first year of life. Moreover, our results lead to clinical reclassification in one single case. Our data emphasize the complex genetic and clinical heterogeneity underlying progeroid disorders

The phenotypic spectrum associated with OTX2 mutations in humans

The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development

Genome-Wide Analysis of Central Corneal Thickness in Primary Open-Angle Glaucoma Cases in the NEIGHBOR and GLAUGEN Consortia

PURPOSE. To investigate the effects of central corneal thickness (CCT)-associated variants on primary open-angle glaucoma (POAG) risk using single nucleotide polymorphisms (SNP) data from the Glaucoma Genes and Environment (GLAUGEN) and National Eye Institute (NEI) Glaucoma Human Genetics Collaboration (NEIGHBOR) consortia. METHODS. A replication analysis of previously reported CCT SNPs was performed in a CCT dataset (n = 1117) and these SNPs were then tested for association with POAG using a larger POAG dataset (n = 6470). Then a CCT genome-wide association study (GWAS) was performed. Top SNPs from this analysis were selected and tested for association with POAG. cDNA libraries from fetal and adult brain and ocular tissue samples were generated and used for candidate gene expression analysis. RESULTS. Association with one of 20 previously published CCT SNPs was replicated: rs12447690, near the ZNF469 gene (P = 0.001; β = −5.08 μm/allele). None of these SNPs were significantly associated with POAG. In the CCT GWAS, no SNPs reached genome-wide significance. After testing 50 candidate SNPs for association with POAG, one SNP was identified, rs7481514 within the neurotrimin (NTM) gene, that was significantly associated with POAG in a low-tension subset (P = 0.00099; Odds Ratio [OR] = 1.28). Additionally, SNPs in the CNTNAP4 gene showed suggestive association with POAG (top SNP = rs1428758; P = 0.018; OR = 0.84). NTM and CNTNAP4 were shown to be expressed in ocular tissues. CONCLUSIONS. The results suggest previously reported CCT loci are not significantly associated with POAG susceptibility. By performing a quantitative analysis of CCT and a subsequent analysis of POAG, SNPs in two cell adhesion molecules, NTM and CNTNAP4, were identified and may increase POAG susceptibility in a subset of cases