PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (50)
 

Clipboard (0)
None

Select a Filter Below

Journals
more »
Year of Publication
1.  Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy 
ACS nano  2013;8(1):546-553.
The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.
doi:10.1021/nn405097u
PMCID: PMC3946819  PMID: 24279711
nanopipette; scanning ion conductance microscopy; single-cell biopsy; electrowetting; mithochondria; next-generation sequencing
2.  Carbohydrate-actuated nanofluidic diode: switchable current rectification in a nanopipette 
Nanoscale  2013;5(19):10.1039/c3nr02105j.
Nanofluidic structures share many properties with ligand-gated ion channels. However, actuating ion conductance in artificial systems is a challenge. We have designed a system that uses a carbohydrate-responsive polymer to modulate ion conductance in a quartz nanopipette. The cationic polymer, a poly(vinylpyridine) quaternized with benzylboronic acid groups, undergoes a transition from swollen to collapsed upon binding to monosaccharides. As a result, the current rectification in nanopipettes can be reversibly switched depending on the concentration of monosaccharides. Such molecular actuation of nanofluidic conductance may be used in novel sensors and drug delivery systems.
doi:10.1039/c3nr02105j
PMCID: PMC3827696  PMID: 23934399
3.  Direct oligonucleotide synthesis onto super-paramagnetic beads 
Journal of biotechnology  2013;167(4):10.1016/j.jbiotec.2013.08.006.
Super-paramagnetic beads (SPMB)s used for a variety of molecular diagnostic assays are prepared by attaching pre-synthesized oligonucleotides to the surface via a cumbersome and low efficient method of carbodiimide-mediated amide bond formation. To mainstream the process, we describe a novel procedure of direct oligonucleotide synthesis onto the surface of SPMBs (e.g. MyOne Dynabeads). With the many challenges surrounding containment of paramagnetic beads (≤ 1 μm) during automated oligonucleotide synthesis, we show that by applying a magnetic force directly to the SPMBs we prevent their loss caused by high-pressure drain steps during synthesis. To date we have synthesized 40mers using a Spacer 9 phosphoramidite (triethylene glycol) coupled to the surface of hydroxylated SPMBs. HPLC analysis shows successful product generation with an average yield of 200 pmoles per sample. Furthermore, because of the versatility of this powerful research tool, we envision its use in any laboratory working with conventional synthesis automation, as employed for single columns and for multi-well titer plates. In addition to direct synthesis of oligodeoxynucleotides (DNA) onto SPMBs, this platform also has the potential for RNA and peptide nucleic acid synthesis.
doi:10.1016/j.jbiotec.2013.08.006
PMCID: PMC3828674  PMID: 23942380
4.  Convergent antibody signatures in human dengue 
Cell host & microbe  2013;13(6):691-700.
SUMMARY
Dengue is the most prevalent mosquito-transmitted viral disease in humans, and the lack of early prognostics, vaccines and therapeutics contributes to immense disease burden. To identify patterns that could be used for sequence-based monitoring of the antibody response to dengue, we examined antibody heavy-chain gene rearrangements in longitudinal peripheral blood samples from 60 dengue patients. Comparing signatures between acute dengue, post-recovery and healthy samples, we find increased expansion of B cell clones in acute dengue patients, with higher overall clonality in secondary infection. Additionally, we observe consistent antibody sequence features in acute dengue in the major antigen-binding determinant Complementarity Determining Region-3 (CDR3), with specific CDR3 sequences highly enriched in acute samples compared to post-recovery, healthy or non-dengue samples. Dengue thus provides a striking example of a human viral infection where convergent immune signatures can be identified in multiple individuals. Such signatures could facilitate surveillance of immunological memory in communities.
doi:10.1016/j.chom.2013.05.008
PMCID: PMC4136508  PMID: 23768493
5.  The Sequencing Bead Array (SBA), a Next-Generation Digital Suspension Array 
PLoS ONE  2013;8(10):e76696.
Here we describe the novel Sequencing Bead Array (SBA), a complete assay for molecular diagnostics and typing applications. SBA is a digital suspension array using Next-Generation Sequencing (NGS), to replace conventional optical readout platforms. The technology allows for reducing the number of instruments required in a laboratory setting, where the same NGS instrument could be employed from whole-genome and targeted sequencing to SBA broad-range biomarker detection and genotyping. As proof-of-concept, a model assay was designed that could distinguish ten Human Papillomavirus (HPV) genotypes associated with cervical cancer progression. SBA was used to genotype 20 cervical tumor samples and, when compared with amplicon pyrosequencing, was able to detect two additional co-infections due to increased sensitivity. We also introduce in-house software Sphix, enabling easy accessibility and interpretation of results. The technology offers a multi-parallel, rapid, robust, and scalable system that is readily adaptable for a multitude of microarray diagnostic and typing applications, e.g. genetic signatures, single nucleotide polymorphisms (SNPs), structural variations, and immunoassays. SBA has the potential to dramatically change the way we perform probe-based applications, and allow for a smooth transition towards the technology offered by genomic sequencing.
doi:10.1371/journal.pone.0076696
PMCID: PMC3792038  PMID: 24116138
6.  Evaluation of ProExC as a Prognostic Marker in Oropharyngeal Squamous Cell Carcinomas 
ProExC expression has been shown to perform similarly to p16 as an aid in the diagnosis of cervical dysplasia but has not been well characterized in head and neck squamous cell carcinomas (SCC). The purpose of this study is to determine if ProExC performs similarly to p16 as a prognostic marker in oropharyngeal SCC and to evaluate the threshold of ProExC and p16 staining that correlates with survival. ProExC, p16 and HPV DNA in situ hybridization (ISH) were performed on tissue microarray (TMA) cores and whole sections from 62 patients with oropharyngeal SCC. Sensitivity and specificity for high-risk HPV and correlation with overall survival (OS), cancer-specific survival (CSS) and time to distant metastasis (TDM) were calculated for ProExC and p16 at different thresholds. ProExC did not prove to be a robust marker. It showed strong correlation with OS at a 66% threshold on TMA cores but correlation with OS was lost on whole sections. It also exhibited low sensitivity (53.7%) on TMA cores and low specificity on whole sections (65%). ProExC at a 33% threshold exhibited unacceptably low specificity and did not correlate with OS, CSS or TDM. Sensitivity and specificity of p16 varied predictably with threshold: higher sensitivity and lower specificity with lower thresholds and vice versa for higher thresholds. p16 at a 50% threshold offers a balance between sensitivity and specificity, and correlates with OS, CSS and TDM on whole sections; correlation with TDM is lost on TMA cores. These findings indicate that ProExC does not perform well enough to be used as a prognostic marker in oropharyngeal SCC. p16 should be used and scored as positive when at least half the tumor is strongly staining.
doi:10.1097/PAS.0b013e3182600eaa
PMCID: PMC3397679  PMID: 22790856
head and neck; squamous cell carcinoma; oropharynx; ProExC; p16; HPV; in situ hybridization; PCR
7.  HLA Haplotyping from RNA-seq Data Using Hierarchical Read Weighting 
PLoS ONE  2013;8(6):e67885.
Correctly matching the HLA haplotypes of donor and recipient is essential to the success of allogenic hematopoietic stem cell transplantation. Current HLA typing methods rely on targeted testing of recognized antigens or sequences. Despite advances in Next Generation Sequencing, general high throughput transcriptome sequencing is currently underutilized for HLA haplotyping due to the central difficulty in aligning sequences within this highly variable region. Here we present the method, HLAforest, that can accurately predict HLA haplotype by hierarchically weighting reads and using an iterative, greedy, top down pruning technique. HLAforest correctly predicts >99% of allele group level (2 digit) haplotypes and 93% of peptide-level (4 digit) haplotypes of the most diverse HLA genes in simulations with read lengths and error rates modeling currently available sequencing technology. The method is very robust to sequencing error and can predict 99% of allele-group level haplotypes with substitution rates as high as 8.8%. When applied to data generated from a trio of cell lines, HLAforest corroborated PCR-based HLA haplotyping methods and accurately predicted 16/18 (89%) major class I genes for a daughter–father-mother trio at the peptide level. Major class II genes were predicted with 100% concordance between the daughter–father-mother trio. In fifty HapMap samples with paired end reads just 37 nucleotides long, HLAforest predicted 96.5% of allele group level HLA haplotypes correctly and 83% of peptide level haplotypes correctly. In sixteen RNAseq samples with limited coverage across HLA genes, HLAforest predicted 97.7% of allele group level haplotypes and 85% of peptide level haplotypes correctly.
doi:10.1371/journal.pone.0067885
PMCID: PMC3696101  PMID: 23840783
8.  Use of Whole Genome Sequencing to Determine the Microevolution of Mycobacterium tuberculosis during an Outbreak 
PLoS ONE  2013;8(3):e58235.
Rationale
Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods.
Objective
To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain.
Method and Measurements
We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina’s sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing.
Main results
We obtained an average of 95.7% (94.1–96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0–2 mutations per transmission event that resulted in a secondary case.
Conclusions
Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0–2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.
doi:10.1371/journal.pone.0058235
PMCID: PMC3589338  PMID: 23472164
9.  Quantitation of the Human Papillomavirus DNA in the Plasma of Patients with Oropharyngeal Carcinoma 
Purpose
To determine whether HPV DNA can be detected in the plasma of patients with HPV(+) oropharyngeal carcinoma (OP) and to monitor its temporal change during radiotherapy (RT).
Methods and Materials
We used PCR to detect HPV DNA in the culture media of HPV(+) SCC90, VU147T and the plasma of SCC90 and HeLa tumor bearing mice, non-tumor controls and those bearing HPV(-) tumors. We used real time quantitative PCR (qPCR) to quantify plasma HPV DNA in 40 HPV(+) OP, 24 HPV(-) head and neck cancer (HNC) patients and 10 non-cancer volunteers. Tumor HPV status was confirmed by p16INK4a staining and HPV16/18 PCR or HPV ISH. 14 patients had serial plasma samples for HPV DNA quantification during RT.
Results
HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in controls. It was detected in 65% of pretreatment plasma samples from HPV(+) OP patients using E6/7 qPCR. None of the HPV(-) HNC or non-cancer controls had detectable HPV DNA. Pretreatment plasma HPV DNA copy number correlated significantly with nodal metabolic tumor volume (assessed on FDG-PET). Serial measurements in 14 patients showed rapid decline in HPV DNA that became undetectable at RT completion. In 3 patients, HPV DNA rose to discernable level at the time of metastasis.
Conclusions
Xenograft studies indicated that plasma HPV DNA is released from HPV(+) tumors. Circulating HPV DNA is detectable in most HPV(+) OP patients. Plasma HPV DNA may be a valuable tool for identifying relapse.
doi:10.1016/j.ijrobp.2011.05.061
PMCID: PMC3257411  PMID: 21985946
Human papillomavirus; oropharyngeal carcinoma; radiotherapy; plasma; circulating DNA
10.  Identification of radiation-induced microRNA transcriptome by next-generation massively parallel sequencing 
Journal of Radiation Research  2013;54(5):808-822.
Gene regulation in cells exposed to ionizing radiation (IR) occurs at the transcriptional and post-transcriptional levels. Recent studies have suggested that micro-RNA (miRNA) play a significant role in post-transcriptional gene regulation in irradiated cells. miRNA are RNA molecules 18–24 nucleotides in length that are involved in negatively regulating the stability or translation of target messenger RNA. Previous studies from our laboratory have shown that the expression of various miRNA is altered in IR-treated cells. In the present study we monitored genome-wide expression changes of miRNA transcriptome by massively parallel sequencing of human cells irradiated with X-rays. The baseline expression of 402 miRNA indicated a wide range of modulation without exposure to IR. Differences in the expression of many miRNA were observed in a time-dependent fashion following radiation treatment. The Short Time-series Expression Miner (STEM) clustering tool was used to characterize 190 miRNA to six statistically significant temporal expression profiles. miR-19b and miR-93 were induced and miR-222, miR-92a, and miR-941 were repressed after radiation treatment. miR-142-3p, miR-142-5p, miR-107, miR-106b, miR-191, miR-21, miR-26a, miR-182, miR-16, miR-146a, miR-22 and miR-30e exhibited two peaks of induction: one at 8 h and the other at 24 h post-irradiation. miR-378, miR-let-7a, miR-let-7g, miR-let-7f, miR-103b, miR-486-3p, miR-423-5p, miR-4448, miR-3607-5p, miR-20b, miR-130b, miR-155, miR-181, miR-30d and miR-378c were induced only at the 8-h time-point. This catalogue of the inventory of miRNA that are modulated as a response to radiation exposure will be useful for explaining the mechanisms of gene regulation under conditions of stress.
doi:10.1093/jrr/rrt014
PMCID: PMC3766286  PMID: 23447695
micro-RNA; differential gene expression; next-generation sequencing; TK6 cells; radiation effects
11.  Copper Sensing with a Prion Protein Modified Nanopipette 
RSC advances  2012;2(31):11638-11640.
Protein-metal interactions determine and regulate many biological functions. Nanopipettes functionalized with peptide moieties can be used as sensors for metal ions in solution.
doi:10.1039/C2RA21730A
PMCID: PMC3519431  PMID: 23243499
13.  Reversible thrombin detection by aptamer functionalized STING sensors 
Biosensors & bioelectronics  2011;26(11):4503-4507.
Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution.
doi:10.1016/j.bios.2011.05.010
PMCID: PMC3120900  PMID: 21636261
14.  Voltage-controlled metal binding on polyelectrolyte functionalized nanopores 
Most of the research in the field of nanopore-based platforms is focused on monitoring ion currents and forces as individual molecules translocate through the nanopore. Molecular gating, however, can occur when target analytes interact with receptors appended to the nanopore surface. Here we show that a solid state nanopore functionalized with polyelectrolytes can reversibly bind metal ions, resulting in a reversible, real-time signal that is concentration dependent. Functionalization of the sensor is based on electrostatic interactions, requires no covalent bond formation, and can be monitored in real time. Furthermore, we demonstrate how the applied voltage can be employed to tune the binding properties of the sensor. The sensor has wide-ranging applications and, its simplest incarnation can be used to study binding thermodynamics using purely electrical measurements with no need for labeling.
doi:10.1021/la2005612
PMCID: PMC3099346  PMID: 21510657
15.  Molecular probe technology detects bacteria without culture 
BMC Microbiology  2012;12:29.
Background
Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. This molecular probe technology does not require growth of the bacteria in culture. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples.
Results
While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together.
Conclusions
Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. The correlations among the assays were excellent.
doi:10.1186/1471-2180-12-29
PMCID: PMC3316761  PMID: 22404909
16.  Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes 
Genome Biology  2012;13(1):415.
The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.
doi:10.1186/gb-2012-13-1-415
PMCID: PMC3334581  PMID: 22293439
Genomics; evolution; Crocodylia; Archosauria; amniote
17.  NanoStriDE: normalization and differential expression analysis of NanoString nCounter data 
BMC Bioinformatics  2011;12:479.
Background
The nCounter analysis system (NanoString Technologies, Seattle, WA) is a technology that enables the digital quantification of multiplexed target RNA molecules using color-coded molecular barcodes and single-molecule imaging. This system gives discrete counts of RNA transcripts and is capable of providing a high level of precision and sensitivity at less than one transcript copy per cell.
Results
We have designed a web application compatible with any modern web browser that accepts the raw count data produced by the NanoString nCounter analysis system, normalizes it according to guidelines provided by NanoString Technologies, performs differential expression analysis on the normalized data, and provides a heatmap of the results from the differential expression analysis.
Conclusion
NanoStriDE allows biologists to take raw data produced by a NanoString nCounter analysis system and easily interpret differential expression analysis of this data represented through a heatmap. NanoStriDE is freely accessible to use on the NanoStriDE website and is available to use under the GPL v2 license.
doi:10.1186/1471-2105-12-479
PMCID: PMC3273488  PMID: 22177214
18.  UltraSensitive Mycotoxin Detection by STING Sensors 
Biosensors & bioelectronics  2010;26(2):333-337.
Signal Transduction by Ion Nano Gating (STING) technology is a label-free biosensor capable of identifying DNA and proteins. Based on a functionalized quartz nanopipette, the STING sensor includes specific recognition elements for analyte discrimination based on size, shape and charge density. A key feature of this technology is that it doesn't require any nanofabrication facility; each nanopipette can be easily, reproducibly, and inexpensively fabricated and tailored at the bench, thus reducing the cost and the turnaround time. Here, we show that STING sensors are capable of the ultrasensitive detection of HT-2 toxin with a detection limit of 100 fg/ml and compare the STING capabilities with respect to conventional sandwich assay techniques.
doi:10.1016/j.bios.2010.08.016
PMCID: PMC2946441  PMID: 20829024
nanopipette; label-free; single-molecule detection; biosensor; nanopore; STING
19.  Whole-transcriptome RNAseq analysis from minute amount of total RNA 
Nucleic Acids Research  2011;39(18):e120.
RNA sequencing approaches to transcriptome analysis require a large amount of input total RNA to yield sufficient mRNA using either poly-A selection or depletion of rRNA. This feature makes it difficult to miniaturize transcriptome analysis for greater efficiency. To address this challenge, we devised and validated a simple procedure for the preparation of whole-transcriptome cDNA libraries from a minute amount (500 pg) of total RNA. We compared a single-sample library prepared by this Ovation® RNA-Seq system with two available methods of mRNA enrichment (TruSeq™ poly-A enrichment and RiboMinus™ rRNA depletion). Using the Ovation® preparation method for a set of eight mouse tissue samples, the RNA sequencing data obtained from two different next-generation sequencing platforms (SOLiD and Illumina Genome Analyzer IIx) yielded negligible rRNA reads (<3.5%) while retaining transcriptome sequencing fidelity. We further validated the Ovation® amplification technique by examining the resulting library complexity, reproducibility, evenness of transcript coverage, 5′ and 3′ bias and platform-specific biases. Notably, in this side-by-side comparison, SOLiD sequencing chemistry is biased toward higher GC content of transcriptome and Illumina Genome analyzer IIx is biased away from neutral to lower GC content of the transcriptomics regions.
doi:10.1093/nar/gkr547
PMCID: PMC3185437  PMID: 21737426
20.  Sensitive Giant Magnetoresistive-based Immunoassay for Multiplex Mycotoxin Detection 
Biosensors & bioelectronics  2009;25(7):1635-1639.
Rapid and multiplexed measurement is vital in the detection of food-borne pathogens. While highly specific and sensitive, traditional immunochemical assays such as enzyme-linked immunosorbent assays (ELISAs) often require expensive read-out equipment (e.g. fluorescent labels) and lack the capability of multiplex detection. By combining the superior specificity of immunoassays with the sensitivity and simplicity of magnetic detection, we have developed a novel multiplex magnetic nanotag-based detection platform for mycotoxins that functions on a sub-picomolar concentration level. Unlike fluorescent labels, magnetic nanotags (MNTs) can be detected with inexpensive giant magnetoresistive (GMR) sensors such as spin-valve sensors. In the system presented here, each spin-valve sensor has an active area of 90 × 90 µm2, arranged in an 8×8 array. Sample is added to the antibody-immobilized sensor array prior to the addition of the biotinylated detection antibody. The sensor response is recorded in real time upon the addition of streptavidin-linked MNTs on the chip. Here we demonstrate the simultaneous detection of multiple mycotoxins (aflatoxins B1, zearalenone and HT-2) and show that a detection limit of 50 pg/mL can be achieved.
doi:10.1016/j.bios.2009.11.028
PMCID: PMC2947153  PMID: 20047828
21.  Detection of point mutations associated with antibiotic resistance in Pseudomonas aeruginosa 
Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing™ for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing™ as a substitute for traditional methods as it provides a rapid and reliable technique for determinating the antibiotic resistance pattern of a given bacterial strain in <1 h.
doi:10.1016/j.ijantimicag.2009.05.013
PMCID: PMC2744841  PMID: 19656662
Pyrosequencing; Pseudomonas aeruginosa; Antibiotic resistance
22.  Pyrosequencing of the DNA gyrase gene in Neisseria species: effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae 
The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.
doi:10.1111/j.1600-0463.2006.apm_495.x
PMCID: PMC2953271  PMID: 17207083
Neisseria gonorrhoeae; Neisseria species; ciprofloxacin; gyrA; species verification
23.  Current Rectification with Poly-l-Lysine-Coated Quartz Nanopipettes 
Nano letters  2006;6(11):2486-2492.
Ion current rectification with quartz nanopipette electrodes was investigated through the control of the surface charge. The presence and absence of a positively charged poly-l-lysine (PLL) coating resulted in the rectified current with opposite polarity. The results agreed with the theories developed for current-rectifying conical nanopores, suggesting the similar underlying mechanism among asymmetric nanostructure in general. This surface condition dependence can be used as the fundamental principle of multi-purpose real-time in vivo biosensors.
doi:10.1021/nl061681k
PMCID: PMC2948113  PMID: 17090078
24.  Template tailoring: Accurate determination of heterozygous alleles using peptide nucleic acid and dideoxyNTP 
Electrophoresis  2010;31(8):1322-1329.
Measurement of the length of DNA fragments plays a pivotal role in genetic mapping, disease diagnostics, human identification and forensic applications. PCR followed by electrophoresis is used for DNA length measurement of STRs, a process that requires labeled primers and allelic ladders as standards to avoid machine error. Sequencing-based approaches can be used for STR analysis to eliminate the requirement of labeled primers and allelic ladder. However, the limiting factor with this approach is unsynchronized polymerization in heterozygous sample analysis, in which alleles with different lengths can lead to imbalanced heterozygote peak height ratios. We have developed a rapid DNA length measurement method using peptide nucleic acid and dideoxy dNTPs to “tailor” DNA templates for accurate sequencing to overcome this hurdle. We also devised an accelerated “dyad” pyrosequencing strategy, such that the combined approach can be used as a faster, more accurate alternative to de novo sequencing. Dyad sequencing interrogates two bases at a time by allowing the polymerase to incorporate two nucleotides to DNA template, cutting the analysis time in half. In addition, for the first time, we show the effect of peptide nucleic acid as a blocking probe to stop polymerization, which is essential to analyze the heterozygous samples by sequencing. This approach provides a new platform for rapid and cost-effective DNA length measurement for STRs and resequencing of small DNA fragments.
doi:10.1002/elps.200900631
PMCID: PMC2947149  PMID: 20408144
Dyad pyrosequencing; Peptide nucleic acid; Pyrosequencing; STRs; Template tailoring
25.  Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses 
Molecular and cellular probes  2006;20(3-4):230-238.
Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific ‘sentinel’ base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.
doi:10.1016/j.mcp.2006.01.002
PMCID: PMC2932960  PMID: 16516439
Human papillomaviruses (HPV); DNA sequencing; Multiple infections; Multiple sequencing primers; Sentinel-base DNA sequencing; Pyrosequencing technology

Results 1-25 (50)