Figure 1 - uploaded by Orla Sheils
Content may be subject to copyright.
Peptide nucleic acid in situ hybridisation (PNA-ISH) telomere repeat assay in metaphase spreads showing signals at chromatid ends.
Source publication
Peptide nucleic acid technology (PNA) has become an extremely useful tool and promises to impact on molecular biology and diagnostics. These synthetic DNA analogues pair with DNA and RNA molecules according to Watson and Crick base pairing rules. This paper describes a sensitive and quick fluorescent in situ hybridisation (ISH) technique to determi...
Context in source publication
Context 1
... FITC labelled peptide nucleic acid probe was used to detect several kilobases of tandem repeats at the end of each chromosome by in situ hybridisation. Using a PNA probe comple- mentary to the telomeric repeat sequences, the telomeres appeared as green/yellow fluorescent spots at the end of both chromatids in the met- aphase spreads (fig 1). In interphase nuclei, up to 96 dots can be visualised as scattered intra- (fig 2). Because it is feasible to detect all chromatid telomere repeats in interphase nuclei, the fluorescent PNA-ISH is a very sensitive and eYcient hybridisation method, superior to other oligonucleotide hybridisation protocols for telomeric repeats. [16][17][18] The best results for hybridisation were achieved when the probe and cells were initially denatured at 80°C for three minutes. Results were also obtained for an initial denaturation step at 65°C. Hybridisation was performed for 30 minutes at 37°C, making this method faster than other PNA-ISH protocols. 14 The decreased hybridisation time results from the enhanced hybridisation properties of the PNA ...
Citations
... The TRF blot gives an estimate on the distribution of telomere length and the average length can be extracted (Kimura et al., 2010). A higher resolution-and chromosome-specific analysis can be done with quantitative fluorescence in situ hybridization (Q-FISH) on metaphase chromosomes ( Figure 3D) (Meyne & Moyzis, 1994;Uhlmann et al., 2000;Poon & Lansdorp, 2001). ...
Cancer cells extend critically short telomeres either by reactivating the reverse transcriptase telomerase or by employing alternative lengthening of telomeres (ALT). The ALT mechanism depends on proteins involved in DNA repair and homologous recombination (HR). High-throughput sequencing is increasingly used to analyze whole genome sequences (WGS) and gene expression in patient samples, and potentially, provides a rich resource of information on ALT. However, in ALT cancers the only recurrent mutations identified so far are in the chromatin remodeler ATRX (alpha thalassemia/ mental retardation syndrome X-linked), the histone chaperone DAXX (death domain associated protein), and the histone variant H3.3. In addition, gene expression signatures for patient stratification into ALTpositive and ALT-negative as well as a systematic approach to identify genes involved in telomere maintenance (TM) and in particular ALT via functional annotation are currently missing. One wellestablished hallmark of ALT is the dislocation of the PML (promyelocytic leukemia) protein to telomeres in ALT-associated PML nuclear bodies (APBs). These colocalizations are reliable biomarkers for ALT-positive tumors, but the functional role of PML during the development of ALT remains elusive. In this thesis, I have addressed the issues raised above by work in three areas: First, the TelNet database was developed as a comprehensive compilation of TM genes. Proteins involved in TM were collected, functionally categorized, and evaluated by applying a significance score. In addition to various search modes, a statistics page was implemented for TM pathway analysis and for prediction of the active TM mechanism (TMM). Second, ALT candidate genes were identified by gene expression analysis using four different approaches and isogenic cellular systems: (i) ALT suppression by HDAC inhibitor SAHA in U2OS cells, (ii) ALT induction by ASF1 depletion in HeLa cells with long telomeres (LT), (iii) reduced ALT induction capacity of the ASF1 depletion in HeLa LT cells by SAHA treatment, and (iv) deletion of EST2 (ever shorter telomeres 2), the telomerase protein subunit, in budding yeast to generate survivors that maintain telomeres by type II recombination, equivalent to the human ALT mechanism. A differential gene expression analysis comparing perturbed cells with the unperturbed control cells revealed a positive correlation of WNT and TGFb signaling with the presence of ALT and on the other hand a negative association of TNF/ NFkB/ MAP kinase signaling. Furthermore, a role as potential ALT enhancers was predicted for KCTD15 and TNNC1. In budding yeast type II survivors, approximately 30 genes showed a relatively small albeit statistically significant change in gene expression as compared to pre-senescent cells. Genes within the iron-regulon were overrepresented among downregulated genes, including FIT1, FIT2, ARN2, and FRE4, indicating stress response. Third, I investigated the functional role of PML in the ALT pathway by recruiting PML to telomeres in cells with and without ALT background. The formation of artificial APBs induced telomere clustering and subsequently increased the abundance of extrachromosomal repeats as an ALT feature in both ALT-positive and ALT-negative cells. The results obtained in this thesis facilitate patient stratification based on deep sequencing data according to their TM mechanism and provide a better understanding of the functional role of APBs for ALT.
... There are three reasons for its success: (i) the neutral backbone of PNA facilitates diffusion across cell membranes and also leads to increased rate and stability of hybridization with DNA and RNA targets in fixed cells; 125 (ii) hybridization can occur at very low ionic strengths, which is advantageous as genomic DNA that has been chemically denatured can re-anneal to each other at high ionic strengths, 126 forbidding the binding of probes; and (iii) PNA probes are highly amenable for attachment of a wide selection of fluorophores, 127 thus greatly simplifying multiplex imaging. The use of PNA probes in FISH is established, extensive and clinically relevant, from the visualization of genes and genetic elements in human chromosomes [128][129][130] to the identification of microbial pathogens in infected cells. [131][132][133] The broad applicability of PNA FISH probes is perhaps exemplified by the fact that they can even diffuse across the highly hydrophobic and dense cell membrane of Mycobacterium sp., 134 which is a feat that cannot be accomplished by the intrinsically charged natural nucleic acid probes. ...
Synthetic genetic polymers, also known as xeno-nucleic acids (XNAs), are chemically modified or synthesized analogues of natural nucleic acids. Initially developed by synthetic chemists to better understand nucleic acids, XNAs have grown rapidly over the last two decades in both diversity and usefulness. Their tailor-made functionalities allow them to overcome perennial problems in using natural nucleic acids in technical applications. In this article, key milestones in XNA research are reviewed through highlighting representative examples. The advantages of using XNAs over natural nucleic acids are discussed. It is hoped that this article will provide a summary of the advances and current understanding of XNAs in addition to their technical applications, serving as an entry point to those who are interested in the synthesis and application of XNAs. Besides interesting results, challenges encountered may inspire researchers to perfect the synthesis of XNAs and tailor their functionalities.
... Considering the chemical and biological stability, PNAs could be used to design gene therapeutic drugs [48,49,50] and other molecular biology and functional genomics [51,52]. Besides, PNA probes are extremely useful in situ hybridization and provide very good chromosome images [53,54,55,56,57,58]. A few early reports have examined the properties of PNAs as both specific [59,60,61] as well as general [62] nucleic acid capture probes. ...
Molecular beacons (MBs) of DNA and RNA have aroused increasing interest because they allow a continuous readout, excellent spatial and temporal resolution to observe in real time. This kind of dual-labeled oligonucleotide probes can differentiate between bound and unbound DNA/RNA in homogenous hybridization with a high signal-to-background ratio in living cells. This review briefly summarizes the different unnatural sugar backbones of oligonucleotides combined with fluorophores that have been employed to sense DNA/RNA. With different probes, we epitomize the fundamental understanding of driving forces and these recognition processes. Moreover, we will introduce a few novel and attractive emerging applications and discuss their advantages and disadvantages. We also highlight several perspective probes in the application of cancer therapeutics.
... Telomere FISH on interphase nuclei was used to compare the relative telomere length of coelomic epithelial cell nuclei from regenerating vs. intact arms of the same animals (n 5 6), dissociated by incubation in CMFSS buffer ( Holm et al., 2008) with 10 mM EDTA. The cells were treated with KCl (0.56% w/v) for 20 min and then washed several times in Carnoy´sCarnoy´s fixative (MeOH:HAc (3:1)), as described by Uhlmann et al. (2000). The nuclei suspension was then finally dropped onto SuperFrosts Plus slides. ...
... Southern blot [44,45] Modified SB Slot blot [46] [47] Hybridization protection assay [48] Fluorescence in situ hybridization [50] PNA-FISH FISH with modified probes TELI-FISH T/C-FISH Fiber-FISH [29,49,51,52] [53] [54] [55] [56,57] Flow cytometry-FISH [58][59][60][61][62][63][64] Modified flow-FISH Multicolor flow-FISH [62,65] [ 66] Primed in situ [67][68][69][70] Dideoxy-PRINS Double strand-PRINS [71,72] [73] ...
... Lansdorp et al. developed a quantitative-FISH by using peptide nucleic acid (PNA) oligonucleotide probes (PNA-FISH) instead of oligonucleotide probe [49]. The PNA probe resulting duplexes are more stable than the DNA/DNA or DNA/RNA duplexes formed by the conventional oligonucleotide probes, because the charged phosphate-deoxyribose backbone is replaced by uncharged repeating N-(2-amino ethyl)-glycine backbone linked by peptide bonds [51]. The digital images were recorded with a CCD camera on a fluorescence microscope, and analyzed quantitatively [29,49]. ...
... The modified oligonucleotide probes in the FISH detection of telomere which previously described are suggested to be applied to PRINS [51]. The high target affinity of 2'-OMe-ribose-and 5-(1-propynyl) pyrimidine-modified probes could be suit for PRINS in the detection of telomere [51]. ...
Human telomeres are composed of long repeating sequences of TTAGGG, associated with a variety of telomere-binding proteins. Its function as an end-protector of chromosomes prevents the chromosome from end-to-end fusion, recombination and degradation. Telomerase acts as reverse transcriptase in the elongation of telomeres, which prevent the loss of telomeres due to the end replication problems. However, telomerase activity is detected at low level in somatic cells and high level in embryonic stem cells and tumor cells. It confers immortality to embryonic stem cells and tumor cells. In most tumor cells, telomeres are extremely short and stable. Telomere length is an important indicator of the telomerase activity in tumor cells and it may be used in the prognosis of malignancy. Thus, the assessment of telomeres length is of great experimental and clinical significance. This review describes the role of telomere and telomerase in cancer pathogenesis and the dynamics of the telomeres length in different cell types. The various methods of measurement of telomeres length, i.e. southern blot, hybridization protection assay, fluorescence in situ hybridization, primed in situ, quantitative PCR and single telomere length analysis are discussed. The principle and comparative evaluation of these methods are reviewed. The detection of G-strand overhang by telomeric-oligonucleotide ligation assay, primer extension/nick translation assay and electron microscopy are briefly discussed.
... In fact, PNA can be used for most techniques where DNA would be suitable, and many more besides. Some common applications are Southern blotting [24], in situ hybridization [25,26], fluorescent in situ hybridization (FISH) [27][28][29][30], plasmon resonance [31,32], microarray systems [33], and affinity purification of nucleic acids [34,35]. PNA is a versatile molecule that can be chemically linked to a wide range of other compounds, such as fluorescein, biotin, fatty acids, and peptides, and also it can be chemically modified with other functional groups in place of the standard ethylene linker, such as cyclohexyl or aminoproline, in order to acquire different binding properties. ...
Peptide nucleic acid (PNA) is a hybrid compound with nucleoside bases linked to a peptide-like amide backbone. PNA is capable of sequence-specific base pairing and forms highly stable double and triple helices with natural nucleic acids (DNA, RNA). PNA forms stable hydrogen bonds and is resistant to degradation by nucleases and proteases. Because of these physico-chemical properties, PNA has attracted great attention, since its first description in 1991, as a potential gene-specific drug and a versatile molecular biology tool. More and more laboratories are working with PNA, and the number of applications in which PNA proves useful continues to increase. In this chapter, we describe aspects of the biochemistry of peptide nucleic acids and their use as a molecular biology reagent, and then focus on the antisense and anti-gene activity of PNA, with special reference to studies of medical interest, in particular in the PML/RARα and the bcl-2 systems.
Research indicate that telomere is relative to certain diseases such as cancer. Telomerase activity, replicative history and genetic stability of the cells are all reflected in telomere length. Since telomerase is known to be activated and have its length changed by certain cancers, assessments of telomere length are important for disease investigation and analysis. We here describe several methods for detecting telomere length.
Peptide nucleic acid (PNA) is a hybrid compound with nucleoside bases linked to a peptide-like amide backbone. PNA is capable
of sequence-specific base pairing and forms highly stable double and triple helices with natural nucleic acids (DNA, RNA).
PNA forms stable hydrogen bonds and is resistant to degradation by nudeases and proteases. Because of these physicochemical
properties, PNA has attracted great attention, since its first description in 1991, as a potential gene-specific drug and
a versatile molecular biology tool. More and more laboratories are working with PNA and the number of applications in which
PNA proves useful continues to increase.
In this chapter, we describe aspects of the biochemistry of peptide nucleic acids and their use as a molecular biology reagent,
and then focus on the antisense and anti-gene activity of PNA, with special reference to studies of medical interest, in particular
in the PML/RARα and the bcl-2 systems.
During the past ten years, the DNA mimic peptide nucleic acid has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and characterisation of nucleic acids. Most of these methods have taken advantage of the very favourable DNA and RNA hybridisation properties of peptide nucleic acids combined with the unique properties and opportunities offered by peptide chemistry. Within the past year, significant progress in in situ hybridisation technology has been achieved, which has resulted, in particular, in reliable and sensitive methods for detection of bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction clamping method have been expanded, and novel ways of exploiting complexes of peptide nucleic acids with double-stranded DNA, such as double duplex invasion complexes and PD loops, have been developed.
In organisms that propagate by agametic cloning, the parental body is the reproductive unit and fitness increases with clonal size, so that colonial metazoans, despite lack of experimental data, have been considered potentially immortal. Using asexual propagation rate as a measure of somatic performance, and telomerase activity and relative telomere length as molecular markers of senescence, old (7-12 years) asexual strains of a colonial ascidian, Diplosoma listerianum, were compared with their recent sexually produced progeny. We report for the first time evidence for long-term molecular senescence in asexual lineages of a metazoan, and that only passage between sexual generations provides total rejuvenation permitting indefinite propagation and growth. Thus, this colonial ascidian has not fully escaped ageing. The possibility of somatic replicative senescence also potentially helps to explain why metazoans, with the capacity for asexual propagation through agametic cloning, commonly undergo cycles of sexual reproduction in the wild.
