Biomedical subjects

Konan Peck

Publications and source records attributed to Konan Peck.

14 recordsLinked to original sources

PubMed2006

Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays.

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.

Hydrophobic and Hydrophilic Interactions↗

PubMed2006

Design of microarray probes for virus identification and detection of emerging viruses at the genus level.

BACKGROUND: Most virus detection methods are geared towards the detection of specific single viruses or just a few known targets, and lack the capability to uncover the novel viruses that cause emerging viral infections. To address this issue, we developed a computational method that identifies the conserved viral sequences at the genus level for all viral genomes available in GenBank, and established a virus probe library. The virus probes are used not only to identify known viruses but also for discerning the genera of emerging or uncharacterized ones. RESULTS: Using the microarray approach, the identity of the virus in a test sample is determined by the signals of both genus and species-specific probes. The genera of emerging and uncharacterized viruses are determined based on hybridization of the viral sequences to the conserved probes for the existing viral genera. A detection and classification procedure to determine the identity of a virus directly from detection signals results in the rapid identification of the virus. CONCLUSION: We have demonstrated the validity and feasibility of the above strategy with a small number of viral samples. The probe design algorithm can be applied to any publicly available viral sequence database. The strategy of using separate genus and species probe sets enables the use of a straightforward virus identity calculation directly based on the hybridization signals. Our virus identification strategy has great potential in the diagnosis of viral infections. The virus genus and specific probe database and the associated summary tables are available at http://genestamp.sinica.edu.tw/virus/index.htm.

Animals↗

PubMed2006

A multivariate prediction model for microarray cross-hybridization.

BACKGROUND: Expression microarray analysis is one of the most popular molecular diagnostic techniques in the post-genomic era. However, this technique faces the fundamental problem of potential cross-hybridization. This is a pervasive problem for both oligonucleotide and cDNA microarrays; it is considered particularly problematic for the latter. No comprehensive multivariate predictive modeling has been performed to understand how multiple variables contribute to (cross-) hybridization. RESULTS: We propose a systematic search strategy using multiple multivariate models [multiple linear regressions, regression trees, and artificial neural network analyses (ANNs)] to select an effective set of predictors for hybridization. We validate this approach on a set of DNA microarrays with cytochrome p450 family genes. The performance of our multiple multivariate models is compared with that of a recently proposed third-order polynomial regression method that uses percent identity as the sole predictor. All multivariate models agree that the 'most contiguous base pairs between probe and target sequences,' rather than percent identity, is the best univariate predictor. The predictive power is improved by inclusion of additional nonlinear effects, in particular target GC content, when regression trees or ANNs are used. CONCLUSION: A systematic multivariate approach is provided to assess the importance of multiple sequence features for hybridization and of relationships among these features. This approach can easily be applied to larger datasets. This will allow future developments of generalized hybridization models that will be able to correct for false-positive cross-hybridization signals in expression experiments.

Algorithms↗

PubMed2005

Prognosis of non-small cell lung cancer patients by detecting circulating cancer cells in the peripheral blood with multiple marker genes.

PURPOSE: Current lung cancer staging and prognosis methods are based on imaging methods, which may not be sensitive enough for early and accurate detection of metastasis. This study aims to validate the use of a panel of markers for circulating cancer cell detection to improve the accuracy of cancer staging, prognosis, and as a rapid assessment of therapeutic response. EXPERIMENTAL DESIGN: We analyzed the National Cancer Institute-Cancer Genome Anatomy Project database to identify potential marker genes for the detection of circulating cancer cells in peripheral blood. Nested real-time quantitative PCR and a scoring method using cancer cell load Lc were employed to correlate the amount of circulating cancer cells with clinical outcomes in 54 non-small cell lung cancer (NSCLC) patients. The Kaplan-Meier method was employed for analysis of prognostic variables. RESULTS: A panel of four marker genes was identified and experimentally validated. With these marker genes, we achieved an overall positive detection rate of 72% for circulating cancer cells in the peripheral blood of NSCLC patients. Patients who had higher Lc values had worse outcomes and shorter survival times. Patients with poor therapeutic response were revealed by positive detection of circulating cancer cells after therapy. The results correlated well with the patients' survival time. CONCLUSION: Circulating cancer cell detection by a panel of markers and the Lc scoring method can supplement the current tumor, node, metastasis staging method for improved prognosis and for rapid assessment of therapeutic response. Together, they may facilitate the design of better therapeutic strategies for the treatment of NSCLC patients.

Aged↗

PubMed2005

Rapid and sensitive detection of multiple genes from the SARS-coronavirus using quantitative RT-PCR with dual systems.

The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS-CoV) in 2003. To detect early SARS-CoV infection, a one-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture-derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS-S, -M, and -N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture-derived SARS-CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS-N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS-S and SARS-M also demonstrated equivalent sensitivity to the commercially available RealArt HPA-Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS-CoV; and there was no cross detection with other coronaviruses and human respiratory tract-associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple-gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS-CoV infection.

Genes, Viral↗

PubMed2005

SARS-CoV infection was from at least two origins in the Taiwan area.

OBJECTIVE: Severe acute respiratory syndrome (SARS) is caused by a new coronavirus. Genomic sequence analysis will provide the molecular epidemiology and help to develop vaccines. METHODS: We developed a rapid method to amplify and sequence the whole SARS-CoV genome from clinical specimens. The technique employed one-step multiplex RT-PCR to amplify the whole SARS-CoV genome, and then nested PCR was performed to amplify a 2-kb region separately. The PCR products were sequenced. RESULTS: We sequenced the genomes of SARS-CoV from 3 clinical specimens obtained in Taiwan. The sequences were similar to those reported by other groups, except that 17 single nucleotide variations and two 2-nucleotide deletions, and a 1-nucleotide deletion were found. All the variations in the clinical specimens did not alter the amino acid sequence. Of these 17 sequenced variants, two loci (positions 26203 and 27812) were segregated together as a specific genotype - T:T or C:C. Phylogenetic analysis showed two major clusters of SARS patients in Taiwan. CONCLUSION: We developed a very economical and rapid method to sequence the whole genome of SARS-CoV, which can avoid cultural influence. From our results, SARS patients in Taiwan may be infected from two different origins.

Adult↗

PubMed2005

Screening CYP3A single nucleotide polymorphisms in a Han Chinese population with a genotyping chip.

Human cytochrome P450 (CYP)3A is a major P450 enzyme found in the liver and gastrointestinal tract. It plays an important role in the metabolism of a wide variety of drugs, some endogenous steroids and harmful environmental contaminants. It has been shown that CYP3A alleles encoding enzymes with little or no activity are largely created by single nucleotide polymorphisms (SNPs) in the sequences of these genes. The most prevalent of these SNPs are often of low allelic frequency, and many are specific to certain ethnic groups. Therefore, an accurate determination of their frequency in any given ethnic population requires investigations involving large sample sizes. A genotyping chip with enzyme-colorimetric detection was developed and used for simultaneous analysis of 22 known CYP3A SNPs in 451 Han Chinese subjects. Following multiplex polymerase chain reaction and allele-specific primer extension labeling, an enzymatic colorimetry detection system was employed to visualize genotype patterns on a nylon membrane. With this robust system, accurate discrimination ratios were obtained, and approximately 9,922 genotypes were determined. We found that the major CYP3A SNPs in the Chinese subjects were CYP3A4*4 (allele frequency 2.4%), CYP3A4*5 (0.7%), CYP3A4*18A (2.7%) and CYP3A5*3C (70.2%). Most of the major CYP3A4 SNPs found in other ethnicities were not found in this study. Using these SNPs, 11 haplotypes were identified. Comparison between present and previous studies shows that CYP3A4*4 and CYP3A4*5 alleles were Chinese-specific. The genotyping chip developed in this study is an efficient, economic and accurate system for screening multiple SNPs in a large population. Application of such technology is expected to be less labor intensive and easier to adapt to specific searches when compared with other methodologies.

Alleles↗

PubMed2004

Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression.

Gene-specific oligonucleotide probes are currently used in microarrays to avoid cross-hybridization of highly similar sequences. We developed an approach to determine the optimal number and length of gene-specific probes for accurate transcriptional profiling studies. The study surveyed probe lengths from 25 to 1000 nt. Long probes yield better signal intensity than short probes. The signal intensity of short probes can be improved by addition of spacers or using higher probe concentration for spotting. We also found that accurate gene expression measurement can be achieved with multiple probes per gene and fewer probes are needed if longer probes rather than shorter probes are used. Based on theoretical considerations that were confirmed experimentally, our results showed that 150mer is the optimal probe length for expression measurement. Gene-specific probes can be identified using a computational approach for 150mer probes and they can be treated like long cDNA probes in terms of the hybridization reaction for high sensitivity detection. Our experimental data also show that probes which do not generate good signal intensity give erroneous expression ratio measurement results. To use microarray probes without experimental validation, gene-specific probes approximately 150mer in length are necessary. However, shorter oligonucleotide probes also work well in gene expression analysis if the probes are validated by experimental selection or if multiple probes per gene are used for expression measurement.

Escherichia coli↗

PubMed2004

Differential gene expression in gram-negative and gram-positive sepsis.

Sepsis is the most common cause of death in patients in the intensive care unit. Genome-wide gene expression analysis can provide insights into the molecular alterations of sepsis. Total mRNA was extracted from the livers of 6 uninfected control mice and 60 septic mice after infusion of either live Escherichia coli or Staphylococcus aureus. Using a murine complementary DNA microarray system, changes in gene expression were monitored at six time points (uninfected, 2, 8, 24, 48, and 72 hours). Overall, 4.8% of 6,144 assessed genes were differentially regulated with a greater than twofold change across all time points. Most of the genes with altered expression were commonly present in gram-negative and gram-positive sepsis, but the expression levels of 17 genes were different between both types of sepsis at particular time points after infection. The microarray results support the hypothesis that both gram-positive and gram-negative sepsis share a final common pathway involved in the pathogenesis of sepsis, but certain genes are differentially expressed under distinct regulation. These results may provide insights into the pathogenesis of sepsis and may also help identify some altered genes that can serve as new targets for diagnostic tools and therapeutic strategies.

Analysis of Variance↗

PubMed2003

Design and assessment of a fast algorithm for identifying specific probes for human and mouse genes.

MOTIVATION: Mammalian genomes are highly complex. To identify the unique sequences of each gene in a mammalian gene database containing tens of thousands of DNA sequences is a computation intensive task. With the advent of parallel genetic analysis methods such as microarrays and the availability of more and more whole genome sequences of organisms, an algorithm allowing speedy identification of the unique gene probes for functional studies of individual genes will be a very useful tool. RESULTS: We have developed a fast algorithm as well as a software program based on the algorithm for identifying gene specific probes of complex organisms. The algorithm was applied to the assemblies of gene sequences and was highly efficient for large databases such as the TIGR human THC and mouse TC databases. The results were assessed with the BLAST sequence alignment software. Two probe data sets have been compiled to contain specific probes for around 100 000 putative human gene transcripts and 70 000 putative mouse gene transcripts. AVAILABILITY: The gene specific probes for the putative human and mouse genes referenced in the TIGR gene indices are available at: ftp://genestamp.ibms.sinica.edu.tw/pub/SpecificP/. The software program and the source codes are available upon request.

Algorithms↗

PubMed2002

Genomic analysis of smooth muscle cells in 3-dimensional collagen matrix.

The proliferation, differentiation, and protein synthesis of vascular smooth muscle cells (SMCs) play important roles in vascular remodeling. Here, we compared the genetic programming and signaling of SMCs in collagen matrix as a three-dimensional (3-D) environment and on a two-dimensional (2-D) surface. By using DNA microarrays with 9600 genes, we showed that 77 genes were expressed more than twofold and 22 genes were less than one-half in 3-D matrix, when compared with the 2-D condition. The higher expression level of cyclin-dependent kinase inhibitor 1 (p21) in 3-D matrix suggests that p21 may be responsible for the lower proliferation rate in 3-D matrix. The expression level of collagen I was higher in 3-D matrix, suggesting that SMCs in 3-D matrix have increased matrix synthesis. In addition, SMCs in 3-D matrix had less stress fibers and focal adhesions, and a lower level of tyrosine phosphorylation of focal adhesion kinase (FAK). Overexpression of FAK attenuated the expression of p21 and collagen I in 3-D matrix, suggesting that FAK functions as a molecular switch for cell cycle regulation and matrix synthesis. The information generated in this study helps to elucidate the molecular basis of the modulation of SMC phenotypes by the extracellular matrix.

Cell Culture Techniques↗

PubMed2002

High throughput parallel synthesis of oligonucleotides with 1536 channel synthesizer.

A 1536 channel oligonucleotide synthesizer, the MultiSyn, was developed with the capability to simultaneously synthesize 1536 oligonucleotides of 20mer length in 10 h. The instrument was designed to synthesize different sequences of various lengths in micro-wells and has synthesized oligonucleotides as long as 119 nt with reasonably good yields using CPG beads of 1000 A pore size. The instrument consists of four 384 channel synthesis modules. Phosphoramidite chemistry was employed and step yields as high as 99.3% were achieved. The enhancement of oligonucleotide synthesis throughput is accomplished by increasing the spatial density of reaction wells. We have identified several parameters that are critical in achieving a good synthesis yield and negligible failure rate in small reaction wells. The coefficient of variation (CV) of product yields in 1536 reaction wells was 20%. The quality of the product was examined by capillary electrophoresis and mass spectrometry. The instrument has robustly synthesized oligonucleotides of various lengths for use as primers and probes for PCR amplifications, oligonucleotide microarrays and genotyping applications. This high throughput oligonucleotide synthesizer is a useful instrument for genomic applications, which require tens of thousands of probes or primers in a short time.

Base Sequence↗

PubMed2002

Matching gene activity with physiological functions.

Matching the activity of the genes with biomechanics and physiology is an effective way to use cDNA microarray technology. Required are data on the change of activities of genes associated with specific physiological functions with respect to a continuous variable such as time. For each pair of data (gene and physiological function) as functions of time, we can compute a coefficient of correlation, R. The correlation is perfect if R is +1 or -1; it is nonexistent if R = 0. By evaluating R for every gene in a microarray, we can arrange the genes in the order of the number R, thus learning which genes are best correlated with the mechanical or physiological function. We illustrate this procedure by studying the blood vessels in the lung in response to pulmonary hypoxic hypertension, including the remodeling of vascular morphometry, the elastic moduli, and the zero-stress state of the vessel wall. For each physiological function, we identify the top genes that correlate the best. We found that different genes correlate best with a given function in large and small arteries, and that the genes in pulmonary veins which respond to arterial functions are different from those in pulmonary arteries. We found one set of genes matching the remodeling of arterial wall thickness, but another set of genes whose integral of activity over time best fit the wall thickness change. Our method can be used to study other thought-provoking problems.

Animals↗

PubMed2002

Changes in gene expression profiles of human fibroblasts in response to sodium arsenite treatment.

Arsenic compounds are widely distributed and arsenic ingestion is associated with many human diseases, including blackfoot disease, atherosclerosis, and cancers. However, the underlying mechanism of arsenic toxicity is not understood. In human fibroblast cells (HFW), arsenite is known to induce oxidative damage, chromosome aberrations, cell cycle arrest, and aneuploidy, and the manifestation of these cellular responses is dependent on changes in gene expression which can be analyzed using the cDNA microarray technique. In this study, cDNA microarray membranes with 568 human genes were used to examine mRNA profile changes in HFW cells treated for 0 to 24 h with 5 microM sodium arsenite. On the basis of the mean value for three independent experiments, 133 target genes were selected for a 2 x 3 self-organizing map cluster analysis; 94 were found to be induced by arsenite treatment, whereas 39 were repressed. These genes were categorized as signal transduction, transcriptional regulation, cell cycle control, stress responses, proteolytic enzymes, and miscellaneous. Significant changes in the signaling-related and transcriptional regulation genes indicated that arsenite induces complex toxicopathological injury.

Arsenites↗