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Provided are a PNA probe for detecting nucleotide polymorphism of a target gene, a melting curve analysis method for detecting the nucleotide polymorphism of the target gene using the same, a nucleotide polymorphism analysis method of a target gene including the melting curve analysis method, and a kit for detecting the nucleotide polymorphism of the target gene containing the PNA probe. It is characterized that the PNA probe according to the present invention contains negative charge molecules. The modified PNA probe according to the present invention contains the negative charge molecules to have a high recognition ability with respect to a target DNA and a high coupling ability to the target DNA and to be rapidly dissociated by heat, such that the nucleotide polymorphism analysis may be relatively easily performed even in a heterozygous sample showing two melting curve graphs, and two or more adjacent single nucleotide polymorphisms may be simultaneously analyzed.

Circulating cell-free DNA (cfDNA) is emerging as a surrogate sample type for mutation analyses. To improve the clinical utility of cfDNA, we developed a sensitive peptide nucleic acid (PNA)-based method for analyzing EGFR and KRAS mutations in the plasma cfDNA of patients with advanced non-small cell lung cancer (NSCLC).Baseline tissue and plasma samples were collected from treatment-nave advanced NSCLC patients participated in a randomized phase II study, which was registered with ClinicalTrials.gov at Feb. 2009 (NCT01003964). EGFR and KRAS mutations in the plasma cfDNA were analyzed retrospectively using a PNA clamping-assisted fluorescence melting curve analysis. The results were compared with those obtained from tissue analysis performed using the direct sequencing. Exploratory analyses were performed to determine survival predicted by the plasma and tissue mutation status.Mutation analyses in matched tissue and plasma samples were available for 194 patients for EGFR and 135 patients for KRAS. The mutation concordance rates were 82.0% (95% confidence interval [CI], 76.5-87.4) for EGFR and 85.9% (95% CI, 80.1-91.8) for KRAS. The plasma EGFR mutation test sensitivity and specificity were 66.7% (95% CI, 60.0-73.3) and 87.4% (95% CI, 82.7-92.1), respectively, and the plasma KRAS mutation test sensitivity and specificity were 50.0% (95% CI, 41.6-58.4) and 89.4% (95% CI, 84.2-94.6), respectively. The predictive value of the plasma EGFR and KRAS mutation status with respect to survival was comparable with that of the tissue mutation status.These data suggest that plasma EGFR and KRAS mutations can be analyzed using PNA-based real-time PCR methods and used as an alternative to tumor genotyping for NSCLC patients when tumor tissue is not available.

Yin H.,University of Oxford | Yin H.,Tianjin Medical University | Betts C.,University of Oxford | Saleh A.F.,Medical Research Council Laboratory of Molecular Biology | And 6 more authors.
Molecular Therapy | Year: 2010

Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. © The American Society of Gene and Cell Therapy.

Jang H.,Panagene Inc. | Kim J.,Panagene Inc. | Choi J.-J.,Panagene Inc. | Son Y.,Panagene Inc. | Park H.,Panagene Inc.
Journal of Clinical Microbiology | Year: 2010

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms. Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Kim J.H.,Korea Research Institute of Bioscience and Biotechnology | Kim J.-W.,Panagene Inc. | Chung B.H.,Korea Research Institute of Bioscience and Biotechnology
Journal of Colloid and Interface Science | Year: 2011

We report an enzymatic method to control the plasmon resonance absorbance of gold nanoparticle (AuNP) arrays assembled on hyaluronic acids. While multiple electrostatic interactions between cysteamine on the AuNPs and the carboxylic acid residues in the whole intact hyaluronic acid induced the formation of large aggregates, precise control of the plasmon absorbance was possible by tailoring the size of the bio-polymeric templates with hyaluronidase, almost over the entire range of the resonant coupling wavelengths. It was possible to precisely tune the position of the second plasmon absorbance by manipulating the amount of the template and the enzymatic hydrolysis time. Finally, we were able to produce a chain-like array of AuNPs, which was nearly one dimensional, with a maximum shift of up to 189. nm in the plasmon absorbance at the optimal hydrolysis time of the templates. This enzymatic method can be used as a useful tool to tailor the plasmonic properties of the nanostructures required for specific applications. © 2011 Elsevier Inc.

Kim H.,Panagene Inc. | Choi J.,Panagene Inc. | Cho M.,Panagene Inc. | Park H.,Panagene Inc.
Biochip Journal | Year: 2012

MicroRNAs (miRNAs) are short, non-coding RNAs that play a critical role in development, metabolism and other fundamental biological processes, and are also important in diseases such as cancer. To study miRNA expression levels in a systematic and parallel manner, we have developed a highly sensitive, specific and reproducible microarray for miRNA expression profiling using the Peptide Nucleic Acids (PNA) probes which have higher affinity and greater specificity for binding to RNA than do DNA probes. We developed a PNA microarray assay that optimizes PNA probe, hybridization conditions, and on-PNA chip labeling method. In this PANArray™ miRNA method, unlabeled RNA is hybridized to the PNA microarray and labeled by enzymatic ligation of pCp-Cy3 on the chip. This assay showed high reproducibility and low cross-hybridization for miRNAs belonging to the let-7 family and the miR-181 family, which differ by a single nucleotide. The PNA microarray (PANArray™ miRNA) is a rapid throughput technology for analyzing expression profiles with high fidelity, and could prove to be a powerful tool for cancer research, diagnosis and prognosic assessment. © 2012 The Korean BioChip Society and Springer-Verlag Berlin Heidelberg.

Oh S.Y.,Panagene Inc. | Ju Y.,Panagene Inc. | Kim S.,Panagene Inc. | Park H.,Panagene Inc.
Oligonucleotides | Year: 2010

MicroRNAs (miRNAs) are noncoding RNAs approximately 22 nucleotides in length that play a major role in the regulation of important biological processes, including cellular development, differentiation, and apoptosis. Antisense oligonucleotides against miRNAs are useful tools for studying the biological mechanisms and therapeutic targets of miRNAs. Various antisense oligonucleotides chemistries, including peptide nucleic acids (PNAs), have been developed to enhance nuclease-resistance and affinity and specificity for miRNA targets. PNAs have a greater specificity and affinity for DNA and RNA than do natural nucleic acids, and they are resistant to nucleases-an essential property of an miRNA inhibitor that will be exposed to cellular nucleases. However, the main limiting factor in the use of PNAs is their reduced penetration into cells. Recently, several cell-penetrating peptides (CPPs) have been investigated as a means to overcome the limited penetration of PNAs. Here, we evaluated the ability of 11 CPPs to transport PNAs inside cells in the absence of transfection reagents and then investigated the ability of these CPPs to inhibit miRNAs. Of the 11 CPPs tested, Tat-modified-conjugated PNA showed the most effective penetration into cells in the absence of transfection reagents and most effectively inhibited miRNAs. Our data demonstrate that Tat-modified-conjugated CPP is the most suitable for supporting PNA-mediated miRNA inhibition. © 2010, Mary Ann Liebert, Inc.

Panagene Inc. | Date: 2010-04-14

This application relates to monomers of the general formula (I) for the preparation PNA (peptide nucleic acid) oligomers and provides method for the synthesis of both predefined sequence PNA oligomers and random sequence PNA oligomers: (I) wherein E is nitrogen or C-R; J is sulfur or oxygen; R, R1, R2, R3, R4 is independently H, halogen, alkyl, nitro, nitrile, alkoxy, halogenated alkyl, halogenated alkoxy, phenyl or halogenated phenyl, R5 is H or protected or unprotected side chain of natural or unnatural a-amino acid; and B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases in the presence of thiol.

Disclosed are a microRNA antisense PNA capable of inhibiting the activity or function of microRNA, a composition for inhibiting the activity or function of microRNA containing the same, a method for inhibiting the activity or function of microRNA using the same, and a method for evaluating the effectiveness thereof.

Panagene Inc. | Date: 2012-11-27

DNA chips; Optical glass for the manufacture of laboratory equipment; Waling glass in the nature of laboratory glassware; lens glass in the nature of eyeglass lenses; ultraviolet-ray transmitting glass in the nature of laboratory glassware; infrared-ray absorbing glass in the nature of laboratory glassware; glass covered with an electrical conductor for use in the manufacture of laboratory glassware; apparatus and instruments for physics, namely, polymerase chain reaction machine, image scanner, spectrophotometer, electrophoresis apparatus for use in laboratories, hybridization chamber, and microarray of biological probes; chemistry apparatus and instruments, namely, pH meter and chromatography apparatus for use in polymer synthesis and purification; and diffraction apparatus, namely, optical lenses and prisms for microscopes. Vaccines and pharmaceutical research and development; Research of geriatric diseases; Bacteriological research; Cancer research; Pharmaceutical development; pharmaceutical research; pharmaceutical testing in the field of pharmacology; chemical-pharmaceutical testing; Medical research, namely, research of medicine, research and development of DNA chips, research of biotechnology; scientific and technical consultation in the field of biotechnology; biological research; Chemical, biochemical, biological and bacteriological research and analysis, namely, analysis of biochemistry, analysis of DNA, and research of genetics; Laboratory apparatus rental; Research and development for others in the fields of microarray; rental of research installations in the nature of laboratory apparatus and instruments; chemistry services, namely, chemical analysis.

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