REVIEW |
The Use of Peptide Nucleic Acids for In Situ Identification of Human Chromosomes
CNRS UPR 1142, Institute of Human Genetics, Montpellier, France (FP); Centre of Assisted Reproduction and Reproductive Genetics, Institute of Biology and Medical Genetics, Motol Hospital, Praha, Czech Republic (PP,MM); and Department of Reproductive Biology B, Arnaud de Villeneuve Hospital, Montpellier, France (SH)
Correspondence to: Dr. Franck Pellestor, CNRS UPR 1142, Institute of Human Genetics, 141 rue de la Cardonille, F-34396 Montpellier Cedex 5, France. E-mail: franck.pellestor{at}igh.cnrs.fr
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Summary |
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Key Words: aneuploidy chromosomes PNA-DNA PNA-FISH synthetic probes
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Introduction |
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Recently, this new type of oligomer has been introduced in cytogenetics. The properties of PNAs have allowed the development of fast, simple, and robust in situ assays, and the efficiency of PNA probes has been demonstrated on various types of cells.
Here we provide an overview of PNA properties and the techniques exploiting PNA technology in molecular genetics and cytogenetics.
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PNA Chemistry and Properties |
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Use of PNAs in Molecular Genetics |
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PNAs as Antigene and Antisense Agents |
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Despite the initial rapid success of PNA-based approaches in vitro, progress in the use of PNAs as tools for regulating gene expression was hampered by the slow cellular uptake of "naked" PNAs by living cells. Subsequent modifications of PNAs have led to significant improvements in the uptake of PNA in eukaryotic cells. The delivery into the cell can be speeded up by coupling PNA to DNA oligomers, to receptor ligands or, more efficiently, to peptides such as liposomes or cell-penetrating peptides that are rapidly internalized by mammalian cells (Pooga et al. 1998; Cutrona et al. 2000
).
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PNA-PCR Strategies |
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The high-affinity binding of PNAs has also been used for detecting single base pair mutations by PCR. This strategy, termed PNA-directed PCR clamping, uses PNAs to inhibit the amplification of a specific target by direct competition of the PNA targeted against one of the PCR primer sites and the conventional PCR primer. This PNA-DNA complex formed at one of the primer sites effectively blocks the formation of the PCR product. The procedure is so powerful that it can be used to detect single base pair gene variants for mutation screening and gene isolation (Orum et al. 1993).
More recently, novel automated real-time PCR has been developed using PNAs. In this method, termed Q-PNA PCR, a generic quencher-labeled PNA (Q-PNA) is hybridized to the 5' tag sequence of a fluorescent dyelabeled DNA primer to quench the fluorescence of the primer. During PCR, the Q-PNA is displaced by incorporation of the primer into amplicons and the fluorescence of the dye label is liberated (Fiandaca et al. 2001).
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Solid-phase Hybridization Techniques |
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The high-affinity binding of PNA oligomers might lead to faster and easier procedures in most standard hybridization techniques, such as Southern and Northern blotting (Nielsen and Egholm 1999). An alternative to Southern blotting analysis is the PNA pre-gel hybridization process, which significantly simplifies the procedure of Southern hybridization. Labeled PNAs are then used as probes, allowing hybridization to a denatured dsDNA sample at low ionic strength before loading on the gel. This is different from conventional Southern blotting, in which hybridization occurs after gel electrophoresis and membrane transfer. Here, the mixture is directly subjected to electrophoresis for separation of bound and unbound PNA probes. Because of their neutral charge, excess unbound PNA probes do not migrate in an electrical field. The PNA-DNA hybrids are then blotted onto a nylon membrane and detected using standard chemiluminescence techniques. The method is sensitive enough to detect a single mismatch in a DNA sample (Perry-O'Keefe et al. 1996
).
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Use of PNAs in Molecular Cytogenetics |
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The PNA-FISH technique was first used for quantitative telomere analysis. The study of telomere behavior has become a sensitive subject because of telomere involvement in the processes of cancer evolution and cellular senescence. The FISH technique has been successfully used for the in situ detection of telomeric repeat sequences in chromosomes of various species, using synthetic oligonucleotide probes, but the efficiency of these probes has not been sufficient to extend this procedure beyond qualitative analysis of repeat telomeric sequences. To monitor telomere length quantitatively, Lansdorp et al. (1996) used fluoresceine-labeled PNA probes. By comparing fluoresceine-labeled DNA, RNA, and PNA probes for the detection of telomeric repeat sequences on human metaphase chromosomes, they first showed that PNA probes yielded superior staining of telomeres. The PNA-FISH approach allowed the distinction of fluorescence of individual sister chromatid ends and the accurate estimate of individual and global telomere length in metaphase chromosomes of various cultured human hematopoietic cells. Subsequently, telomere PNA probes were used in several in situ studies of cancer and aging (Zijlman et al. 1997
; Boei et al. 2000
; Mathioudakis et al. 2000
). The performance of the PNA method for in situ detection and sizing of telomeric repetitive sequences was compared with the primed in situ (PRINS) labeling technique. The two techniques were compared on mouse, hamster, and human cell lines, and the results were identical in terms of labeling efficiency and sensitivity (Serakinci and Koch 1999
).
Further developments of PNA technology were focused on the improvement of the specificity of PNA probes and the in situ detection of numerical chromosome abnormalities. Chen et al. (1999) reported that PNA probes could discriminate between two centromeric DNA repeat sequences that differed by only a single base pair. Identical results were obtained with PRINS primers (Pellestor et al. 1995
) and oligonucleotide probes (O'Keefe et al. 1996
) but never with standard DNA probes. The identification of chromosomal variation and the analysis of polymorphisms could greatly benefit from the discriminative power of PNAs. The procedure of PNA synthesis allows consideration of the further production of allele-specific probes. This will constitute a marked improvement over the current labeling techniques.
Several chromosome-specific PNA probes have been designed and tested. Chen et al. (2000) defined short (1518-mer) and specific PNA probes for alpha-satellite domains of nine chromosomes (chromosomes 1, 2, 7, 9, 11, 17, 18, X, and Y) and successfully used them on metaphase and interphase nuclei. To demonstrate the potential utility of PNA probes in clinical application, cultured and uncultured amniocyte preparations have also been analyzed, giving rates of hybridization efficiency of 9097%. Taneja et al. (2001)
tested other PNA probes for chromosomes 1, X, and Y, 1822-mer in size and directly labeled with fluorochromes, on normal human lymphocytes and fibroblasts with abnormal chromosome contents. A fast and simple multicolor PNA protocol was used, demonstrating the easy use of PNA probes for in situ labeling assays.
Recently, Pellestor et al. (2003) experimented with PNA technology on human sperm. The adaptation of PNA technology to human spermatozoa constituted an interesting challenge because of the pecularities of sperm nuclei in terms of genomic compaction and accessibility of DNA sequences. To estimate and validate the efficiency of PNA labeling on human sperm, comparative estimates of disomies X, Y, and 1 were performed on sperm preparations from healthy subjects using multicolor FISH, PRINS, and PNA procedures in parallel. An equivalent quality of in situ nuclear labeling and similar disomy rates were obtained with the three methods. However, the hybridization timing of PNA probes (i.e., 45 min) was considerably shortened in comparison with FISH reaction, which requires an overnight hybridization to be efficiently completed on sperm preparations. The fast hybridization kinetics of PNAs on sperm were comparable to the kinetics of the PRINS reaction (2030 min). This similarity might be due to the small size of both PNA probes and PRINS primers. These data pointed out the great potential of PNA probes for chromosomal screening on difficult biological material.
Finally, the PNA strategy has been used on isolated human oocytes, polar bodies, and blastomeres to assess the possibility of using PNA probes for preimplantation cytogenetic diagnosis (Paulasova et al. 2004). Using directly labeled satellite PNA probes for chromosomes 1, 4, 9, 16, 18, X, and Y, simple and sequential multicolor PNA labeling procedures were tested on 34 in vitro unfertilized oocytes and 23 blastomeres. The combined use of PNA and FISH was also investigated. Both rates and types of chromosomal abnormalities scored were in good agreement with results of previous FISH studies. This first use of PNA probes on isolated cells confirms the efficiency of PNA technology for in situ chromosomal analysis and demonstrates the feasibility of using PNAs on unique cells. This procedure could become an efficient complement to FISH for preimplantation genetic diagnosis because of its simplicity, its fast kinetics, and the high affinity of PNA probes.
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Conclusion |
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New chemical modifications of the original PNA backbone may contribute to increasing the efficiency of PNA molecules and developing novel applications. Interesting new contributions of PNAs could come from the development of applications in the growing area of whole-genome analysis. The remarkable hybridization properties of PNA suggest that PNA oligomers may be efficiently incorporated into microarrays (Weiler et al. 1997). In association with different fluorochromes, short PNA sequences can constitute a new class of genomic biomarker for microarray platforms and contribute to the next challenge of extending microarray technology to the single-cell level and preimplantation diagnosis (Bermudez et al. 2004
).
Another promising feature of PNAs might be linked to the development of in vivo fluorescence imaging. The capability of introducing fluorescent probes into living cells will allow deeper study of live gene expression and mRNA transfer (Tyagi and Kramer 1996). Because of their high in vivo stability and resistance to enzymes, PNA oligomers conjugated to cell-permeable peptides or liposomes have great potential for contributing to the future of noninvasive medical imaging.
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Acknowledgments |
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Footnotes |
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Received for publication April 5, 2004; accepted August 5, 2004
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