Sensitive and Rapid Detection of Centromeric Alphoid DNA in Human Metaphase Chromosomes by PNA Fluorescence &lt;i&gt;In Situ&lt;/i&gt; Hybridization and Its Application to Biological Radiation Dosimetry

Suto, Yumiko; Hirai, Momoki; Akiyama, Miho; Suzuki, Toshikazu; Sugiura, Norío

doi:10.1508/cytologia.77.261

Cited by 12 publications

(6 citation statements)

References 34 publications

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“…After air-drying, FISH was performed using PNA probes (Cy-3 conjugated human centromeric and FAMconjugated telomere probes; Panagene, Daejon, Korea) (Suto et al 2012). Then the slides were kept in an oven at 64°C for 30 min, treated with 4% paraformaldehide at 4°C for 2 min, rinsed in phosphatebuffered saline (PBS) solution two times at room temperature, dehydrated with an ethanol series (70%, 95% and 99.5%, 1-2 min each), and air-dried.…”

Section: Cytogenetic Procedures For Pccmentioning

confidence: 99%

Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry

Suto¹,

Gotoh²,

Noda³

et al. 2015

Health Physics

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The dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens.

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Section: Cytogenetic Procedures For Pccmentioning

confidence: 99%

Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry

Suto¹,

Gotoh²,

Noda³

et al. 2015

Health Physics

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“…FISH using peptide nucleic acid (PNA) probes (PNA-FISH) was conducted as described previously (Suto et al 2012), with modifications for PCC detection. Chromosome preparations were dried in an oven for 30 min at 64 C, fixed in 4% paraformaldehyde solution at 4 C for 2 min, followed by washing in phosphate buffered saline (PBS) and dehydration with an ethanol series.…”

Section: Fluorescence In Situ Hybridizationmentioning

confidence: 99%

A Modified Protocol for Accurate Detection of Cell Fusion-Mediated Premature Chromosome Condensation in Human Peripheral Blood Lymphocytes

Suto¹,

Akiyama²,

Gotoh³

et al. 2013

CYTOLOGIA

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Summary When interphase cells are fused with mitotic cells, loosely distributing chromatins in nuclei are induced to form prematurely condensed chromosomes. In this paper we report a modified protocol to unequivocally detect prematurely condensed chromosomes in human peripheral blood lymphocytes that were fused with mitotic Chinese hamster ovary (CHO) cells. To examine cell fusion-mediated premature chromosome condensation (PCC), we conducted morphological analysis by differential interference contrast microscopy and molecular cytogenetic analysis by fluorescence in situ hybridization using pan-centromeric and telomeric peptide nucleic acid (PNA) probes. These modified procedures may serve to improve the usefulness of the technique of PCC in cytogenetic investigations.

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“…Labeling of DNA probes for FISH has been generally performed through enzymatic incorporation of labeled dNTPs by PCR or nick translation (Shibata and Hizume 2015). Thyramide signal amplification and peptide nucleic acid probes have contributed to the enhancement of FISH efficiency of low or single-copy genes (Zwirglmaier 2005, Suto et al 2012. However, the method of FISH probe preparation has room for technical improvement.…”

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confidence: 99%

FISH with Padlock Probes Can Efficiently Reveal the Genomic Position of Low or Single-Copy DNA Sequences

Matsunaga

2017

CYTOLOGIA

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A padlock probe is a circularized oligonucleotide containing complementary sequences of target regions. Padlock probes combined with rolling-circle amplification enable fluorescence in situ hybridization (FISH) to reproducibly detect the genomic position of low or single-copy DNA sequences. The FISH with padlock probes revealed that a plant gene cluster of tandemly arrayed three paralogues responsible for photosynthesis rapidly moved from the interior subnuclear region to the peripheral subnuclear region in response to light. Using a pair of peptide nucleic acids as a double-strand opener for the target region, the efficiency of FISH with padlock probes at the detection of a single-copy genomic region was much improved. FISH with padlock probes will give us reliable data of dynamics, arrangement and position of a specific single locus in both the nucleus and the condensed chromosomes.

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Sensitive and Rapid Detection of Centromeric Alphoid DNA in Human Metaphase Chromosomes by PNA Fluorescence <i>In Situ</i> Hybridization and Its Application to Biological Radiation Dosimetry

Cited by 12 publications

References 34 publications

Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry

Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry

A Modified Protocol for Accurate Detection of Cell Fusion-Mediated Premature Chromosome Condensation in Human Peripheral Blood Lymphocytes

FISH with Padlock Probes Can Efficiently Reveal the Genomic Position of Low or Single-Copy DNA Sequences

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