Imaging analysis of EGFR mutated cancer cells using peptide nucleic acid (PNA)–DNA probes

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0–20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

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Quantitative single-nucleotide polymorphism analysis in secondary-structured DNA by affinity capillary electrophoresis using a polyethylene glycol–peptide nucleic acid block copolymer

Analytical Biochemistry ◽

10.1016/j.ab.2012.10.023 ◽

2013 ◽

Vol 433 (2) ◽

pp. 150-152 ◽

Cited By ~ 7

Author(s):

Harumi Tsukada ◽

Lal Mohan Kundu ◽

Yukiharu Matsuoka ◽

Naoki Kanayama ◽

Tohru Takarada ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Capillary Electrophoresis ◽

Polyethylene Glycol ◽

Nucleic Acid ◽

Block Copolymer ◽

Peptide Nucleic Acid ◽

Polymorphism Analysis ◽

Single Nucleotide Polymorphism Analysis ◽

Nucleotide Polymorphism ◽

Single Nucleotide

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Application of a lanthanide fluorescent chelate label for detection of single-nucleotide mutations with peptide nucleic acid probes

Analytical Biochemistry ◽

10.1016/j.ab.2006.06.003 ◽

2006 ◽

Vol 355 (2) ◽

pp. 278-284 ◽

Cited By ~ 12

Author(s):

Kimikazu Hashino ◽

Masahiro Ito ◽

Keisuke Ikawa ◽

Chihiro Hosoya ◽

Takuya Nishioka ◽

...

Keyword(s):

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Single Nucleotide ◽

Nucleic Acid Probes

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Design and Evaluation of Peptide Nucleic Acid Probes for Specific Identification of Candida albicans

Journal of Clinical Microbiology ◽

10.1128/jcm.02417-14 ◽

2014 ◽

Vol 53 (2) ◽

pp. 511-521 ◽

Cited By ~ 8

Author(s):

Hyun-Joong Kim ◽

Byron F. Brehm-Stecher

Keyword(s):

Candida Albicans ◽

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Fungal Infections ◽

Dna Probes ◽

28S Rrna ◽

Rapid Diagnostics ◽

Discriminative Ability ◽

Content Type ◽

Specific Identification

Candida albicansis an important cause of systemic fungal infections, and rapid diagnostics for identifying and differentiatingC. albicansfrom otherCandidaspecies are critical for the timely application of appropriate antimicrobial therapy, improved patient outcomes, and pharmaceutical cost savings. In this work, two 28S rRNA-directed peptide nucleic acid-fluorescencein situhybridization (PNA-FISH) probes, P-Ca726 (targeting a novel region of the ribosome) and P-CalB2208 (targeting a previously reported region), were evaluated. Hybridization conditions were optimized by using both fluorescence microscopy (FM) and flow cytometry (FCM), and probes were screened for specificity and discriminative ability against a panel ofC. albicansand various nontargetCandidaspp. The performance of these PNA probes was compared quantitatively against that of DNA probes or DNA probe/helper combinations directed against the same target regions. Ratiometric analyses of FCM results indicated that both the hybridization quality and yield of the PNA probes were higher than those of the DNA probes. In FCM-based comparisons of the PNA probes, P-Ca726 was found to be highly specific, showing 2.5- to 5.5-fold-higher discriminatory power forC. albicansthan P-CalB2208. The use of formamide further improved the performance of the new probe. Our results reinforce the significant practical and diagnostic advantages of PNA probes over their DNA counterparts for FISH and indicate that P-Ca726 may be used advantageously for the rapid and specific identification ofC. albicansin clinical and related applications, especially when combined with FCM.

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