The peptide nucleic acids, efficient tools for molecular diagnosis (Review)

Pellestor, Franck; Paulasova, Pétra

doi:10.3892/ijmm.13.4.521

Cited by 28 publications

(29 citation statements)

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“…PNAs are synthetic DNA analogs in which the deoxyribose phosphate backbone is replaced by an uncharged polyamid backbone, resulting in a high affinity and specificity to nucleic acids (28). Because PNAs lack a 5Ј hydroxyl moiety, they are resistant to the 5Ј nuclease activity of Taq polymerase and thus are not digested during DNA amplification (4).…”

Section: Discussionmentioning

confidence: 99%

Detection and Discovery of Crustacean Parasites in Blue Crabs ( Callinectes sapidus ) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

Troedsson

Lee

Walters

et al. 2008

Appl Environ Microbiol

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Recently, we described a novel denaturing high-performance liquid chromatography (DHPLC) approach useful for initial detection and identification of crustacean parasites. Because this approach utilizes general primers targeted to conserved regions of the 18S rRNA gene, a priori genetic sequence information on eukaryotic parasites is not required. This distinction provides a significant advantage over specifically targeted PCR assays that do not allow for the detection of unknown or unsuspected parasites. However, initial field evaluations of the DHPLC assay suggested that because of PCR-biased amplification of dominant host genes it was not possible to detect relatively rare parasite genes in infected crab tissue. Here, we describe the use of a peptide nucleic acid (PNA) PCR hybridization blocking probe in association with DHPLC (PNA-PCR DHPLC) to overcome inherent PCR bias associated with amplification of rare target genes by use of generic primers. This approach was utilized to detect infection of blue crabs (Callinectes sapidus) by the parasitic dinoflagellate Hematodinium sp. Evaluation of 76 crabs caught in Wassaw Sound, GA, indicated a 97% correspondence between detection of the parasite by use of a specific PCR diagnostic assay and that by use of PNA-PCR DHPLC. During these studies, we discovered one crab with an association with a previously undescribed protist symbiont. Phylogenetic analysis of the amplified symbiont 18S rRNA gene indicated that it is most closely related to the free-living kinetoplastid parasite Procryptobia sorokini. To our knowledge, this is the first report of this parasite group in a decapod crab and of this organism exhibiting a presumably parasitic life history.Parasites, parasitoids, and infectious diseases are recognized as significant biotic factors affecting individuals, populations, communities, and ecosystems and have been implicated in global-scale declines of a wide range of marine and terrestrial species (11,17,22,25,27). For example, the world's coral reefs are currently declining at an alarming rate and although the role of disease is clearly prominent, many of the responsible disease agents remain unknown (12,32,36). Historically, discovery and investigation of disease processes have relied exclusively on labor-intensive histological and culture-based techniques. Methodological limitations have therefore been recognized as a major impediment to the study of emerging diseases in marine species. The lack of knowledge about the existence and biology of many parasites and parasitoids limits our ability to recognize the emergence of new diseases and to identify complex life histories and vectors involved in disease etiology. These difficulties are particularly acute for those trying to understand linkages between environmental stressors and disease since such studies demand the analysis of large numbers and diversities of samples (21).During the past decade, new molecular diagnostic techniques have enabled detection and quantification of a large range of parasites, parasit...

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Section: Discussionmentioning

confidence: 99%

Detection and Discovery of Crustacean Parasites in Blue Crabs ( Callinectes sapidus ) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

Troedsson

Lee

Walters

et al. 2008

Appl Environ Microbiol

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“…Because PNAs increase T m values by approximately 1.0°C per base pair for a PNA-DNA duplex and ⌬T m values of PNA-DNA duplexes are 2.5 to 8.5°C higher than those of the corresponding DNA-DNA duplexes, PNA probes are expected to be more accurate. The following factors probably contribute to the higher sensitivity of these arrays: (i) improved detection of sequence variations by novel PNA probes, (ii) improved detection limits of PNA probes, and (iii) overall higher sensitivity of PNA arrays (3,18). PNA-DNA hybrids offer greater stability than their DNA-DNA or DNA-RNA counterparts.…”

Section: Discussionmentioning

confidence: 99%

“…The synthetic backbone of PNAs also means that they are not degraded by nucleases or proteases. Because of this resistance to enzyme degradation, the lifetime of PNAs is extended both in vivo and in vitro (18). The PNA arrays were found to have a shelf life longer than 13 months when stored in slide boxes at room temperature (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The particular biochemical properties of PNA make it suitable for use in many biological applications. An increasing number of applications for PNA technology have been described, confirming the high potential of PNAs as probes for antisense, PCR clamping, fluorescence in situ hybridization, and microarrays (2,12,18). The purpose of this study was to develop a PNA array for the genotyping of 32 medically important HPVs and to evaluate the PANArray HPV (for research use only, not for use in diagnostic procedures) using clinical samples.…”

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confidence: 91%

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Peptide Nucleic Acid-Based Array for Detecting and Genotyping Human Papillomaviruses

Choi¹,

Kim²,

Park³

2009

J Clin Microbiol

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We describe a novel array for accurate and reliable genotyping of human papillomavirus (HPV) using peptide nucleic acid (PNA) probes. In order to exploit the superior hybridization properties of PNA with target HPV DNAs, we developed a novel PNA array (PANArray HPV). PANArray HPV enables the detection and genotyping of HPVs using 32 type-specific PNA capture probes for medically important HPVs. All tested HPV types showed highly unique hybridization patterns with type-specific PNA probes. PNA array results showed stable specificities and sensitivities after up to 13 months of storage at room temperature. Also, we demonstrated the superior specificity, sensitivity, and stability of PNA arrays for HPV genotyping. We compared the genotyping results of the PNA array to sequencing with MY09/11 PCR products derived from 72 clinical samples. The results showed excellent agreement between the PNA array and sequencing, except for samples reflecting multiple infections. The results from the PNA array were compared with those of type-specific PCR when discrepant results occurred owing to multiple infections. The results for the PNA array matched those of type-specific PCR in all cases. Newly developed PNA arrays show excellent specificity and sensitivity and long shelf life. Our results suggest that the PNA array represents a reliable alternative to conventional DNA arrays for HPV genotyping, as well as for diagnostics.Human papillomaviruses (HPVs) comprise more than 100 genotypes, of which over 30 types are associated with sexually transmitted infections. The mucosal types can be divided into high-risk and low-risk types depending on the associated disease risk. HPV infection is a necessary prerequisite for cervical cancer development. Accurate HPV genotyping is increasingly important for investigating the clinical outcomes and epidemiologies of particular types, for the characterization of study populations in HPV vaccination trials, and for monitoring the efficacies of HPV vaccines (17,20).Members of the HPV family do not lend themselves to culture in vitro. Thus, detection of HPV is restricted to molecular analyses of HPV DNA sequences (10). Technical improvements in PCR, sequencing, and hybridization assays (ThirdWave Technology hybrid capture, linear array, and oligonucleotide arrays) have increased the sensitivity, specificity, and speed of assays. Although this technology has many advantages, and several working systems have been established, their sensitivity, reproducibility, and reliability are still major issues (6,8,11,21,26). Most platforms that have been described make use of natural DNA or RNA as capture probes. However, several nucleic acid analogs have demonstrated more favorable hybridization characteristics than standard DNA-based probes. Nucleic acid analogs have been made in order to overcome the limitations of natural nucleic acids for specificity, sensitivity, hybridization kinetics, thermodynamic properties, and stability (2,4,12).Peptide nucleic acids (PNAs) are notable for their exceptional biolo...

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“…PNAs are highly resistant to in vivo cleavage. Interested readers are referred to several excellent reviews for more detailed information on oligonucleotide modifications, mechanisms of action and their potential applications in medicine (Urban and Noe, 2003;Buchini and Leumann, 2003;Kurreck, 2003, Kaihatsu et al, 2004Pellestor and Paulasova, 2004).…”

Section: Oligonucleotides and Nucleic Acidsmentioning

confidence: 99%

Applications of nucleoside-based molecular probes for the in vivo assessment of tumour biochemistry using positron emission tomography (PET)

Wiebe¹

2007

Braz. arch. biol. technol.

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Positron emission tomography (PET) is a non-invasive nuclear imaging technique. In PET, radiolabelled molecules decay by positron emission. The gamma rays resulting from positron annihilation are detected in coincidence and mapped to produce three dimensional images of radiotracer distribution in the body. Molecular imaging with PET refers to the use of positron-emitting biomolecules that are highly specific substrates for target enzymes, transport proteins or receptor proteins. Molecular imaging with PET produces spatial and temporal maps of the targetrelated processes. Molecular imaging is an important analytical tool in diagnostic medical imaging, therapy monitoring and the development of new drugs. Molecular imaging has its roots in molecular biology

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The peptide nucleic acids, efficient tools for molecular diagnosis (Review)

Cited by 28 publications

References 0 publications

Detection and Discovery of Crustacean Parasites in Blue Crabs ( Callinectes sapidus ) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

Detection and Discovery of Crustacean Parasites in Blue Crabs ( Callinectes sapidus ) by Using 18S rRNA Gene-Targeted Denaturing High-Performance Liquid Chromatography

Peptide Nucleic Acid-Based Array for Detecting and Genotyping Human Papillomaviruses

Applications of nucleoside-based molecular probes for the in vivo assessment of tumour biochemistry using positron emission tomography (PET)

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