16

Peptide : a versatile tool in genetic diagnostics and Peter E Nielsen

During the past ten years, the DNA mimic nucleic acid Figure 1 has inspired the development of a variety of hybridisation-based methods for detection, quantification, purification and B characterisation of nucleic acids. Most of these methods have B B taken advantage of the very favourable DNA and RNA O hybridisation properties of peptide nucleic acids combined with OÐ O O the unique properties and opportunities offered by peptide O O OÐ P O O chemistry. Within the past year, significant progress in in situ P (5′) O O O hybridisation technology has been achieved, which has resulted, O in particular, in reliable and sensitive methods for detection of DNA bacteria in clinical samples, as well as in environmental samples. Furthermore, applications of the polymerase chain reaction B B clamping method have been expanded, and novel ways of B exploiting complexes of peptide nucleic acids with double- O O O stranded DNA, such as double duplex invasion complexes and N N N NH PD loops, have been developed. NH NH NH

O Address (Amino-terminal) O O Center for Biomolecular Recognition, Department for Biochemistry PNA and Genetics, Laboratory B, The Panum Institute, Blegdamsvej 3c, DK-2200 N Copenhagen, Denmark Current Opinion in Biotechnology

Current Opinion in Biotechnology 2001, 12:16–20 Chemical structures of PNA and DNA.

0958-1669/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. × Tmpred = 20.79 + 0.83 TmDNA Abbreviations –26.13 × f + 0.44 × length (1) ds double-stranded pyr PNA Tm thermal stability Where TmDNA is the predicted Tm value for the analo- gous DNA–DNA duplex according to SantaLucia et al. [4] (not including end effects), and fpyr is the con- Introduction tent (or fraction) of the PNA strand. It was clear, even on the introduction of peptide nucleic acids (PNAs) in 1991 [1], that this DNA mimic (see Figure 1) Notably, these data indicate that at physiological ionic could play a role in improving existing and developing novel strength, mixed-sequence PNA–DNA duplexes are, in techniques within DNA hybridisation-based methods in general, slightly more stable (ca. 1°C/bp) than the genetic diagnostics and molecular biology. It often has been corresponding DNA–DNA duplexes. However, pyrimi- argued that PNA hybridises more strongly to complementary dine-rich PNA–DNA duplexes are less stable, and DNA and RNA than do natural , but -rich PNA–DNA duplexes are much more stable with better sequence discrimination; therefore, any hybridi- than their DNA–DNA counterparts. Furthermore, sation-based technique employing PNA probes instead of homopyrimidine PNAs are exceptional in that they form DNA probes should perform better. This is not correct for extremely stable PNA–DNA triplexes containing two several reasons. First, the hybridisation strength — PNA strands. Therefore, careful sequence considera- expressed as the thermal stability (Tm) for PNA–DNA (and tions are mandatory when designing a PNA probe for use presumably also PNA–RNA) duplexes — display a much in a hybridisation experiment. more complex sequence dependence compared with DNA–DNA duplexes. A PNA–DNA duplex is not symmet- Second, it should be kept in mind that PNA is a non- rical and the Tm shows extreme variation that is dependent charged pseudopeptide that has physico-chemical on the purine content of the PNA strand [2]. This effect is properties that differ significantly from polyanionic additional to the sequence dependence observed for the sta- . Therefore, any experimental conditions bility of DNA–DNA duplexes, and an empirical formula for in hybridisation or other assays that have been optimised calculating the Tm of a PNA–DNA duplex (+/–5°C has been for oligonucleotides cannot be expected to be optimal, or derived [3] and is shown in Equation 1: even suitable, when employing PNAs. Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology Nielsen 17

Figure 2

Schematic representation of various complexes formed by PNA binding to duplex (a) (b) (c) (d) DNA. The ladder symbolises the DNA double helix, and the PNA is drawn in bold. (a) Conventional triplex and (b) triplex invasion complexes are formed at homopurine DNA targets with complementary homopyrimidine PNAs. The triplex invasion complexes have extraordinary stability, whereas conventional triplexes have been observed in a few cases and are far less stable than the corresponding triplex invasion complexes. (c) Duplex invasion complexes are formed with some homopurine PNAs are fairly unstable, whereas double duplex invasion complexes (d) are very stable and require modified pairs such as Triplex Triplex invasion Duplex invasion Double duplex invasion diaminopurine–thiouracil. Current Opinion in Biotechnology

It also is important to note that the stability of PNA–DNA giving very good signal-to-noise ratios and also allowing (or PNA–RNA) complexes (which is due to the non- employment of hybridisation and washing procedures that charged PNA backbone) is virtually insensitive to ionic yield good chromosome images [13–19]. Of particular strength [5,6]. This contrasts with the stability of interest, telomeric PNA probes are now being used DNA–DNA or RNA–RNA duplexes, which are signifi- increasingly in cancer and ageing research, and recently, in cantly destabilised at very low ionic strength. This combination with centromeric DNA probes to study X-ray- property of PNAs can be exploited when targeting DNA or induced chromosome exchange [18,19]. Further RNA sequences that are involved in or have a propensity developments have resulted in the identification, so far, of to form a secondary structure [7]. ten chromosome-specific PNA probes (for chromosomes 1, 2, 7, 9, 11, 17, 18, X and Y) which utilise satellite repeat Targeting of double-stranded (ds) DNA with PNA can occur sequences. Such probes may become useful for diagnosis via at least four different binding modes (Figure 2). Three of of chromosomal anomalies [20•]. these modes (e.g. triplex formation, duplex invasion and triplex invasion) require homopurine/homopyrimidine DNA PNA–FISH techniques also were developed recently for targets, whereas double duplex invasion (i.e. exploiting detection and identification of bacteria in medical diagnos- pseudocomplementary PNA containing 2,4- tics [19–23,24•] and in environmental analysis (for diaminopurine and 4-thiouracil instead of adenine (A) and example, water pollution) [25•]. In these cases, PNA thymine (T), respectively [8••]) requires targets of at least probes specific for sequences of the ribosomal RNA of the 50% AT content. For practical applications, the triplex and microorganism are employed [23,24•] and (quantitative) double duplex invasion complexes with targets of at least 8 bp analysis was performed by fluorescence microscopy or by will have sufficient stability. It is also important to note that laser scanning of filtered bacteria. This technique is very the formation of such invasion complexes is very slow at ele- fast and has good sensitivity, but does not readily distin- vated ionic strength (i.e. >50 mM Na+/K+) or in the presence guish naturally between live and dead bacteria. of divalent or multivalent cations (e.g. Mg2+ and spermine). Therefore, binding reactions are performed most convenient- Nucleic acid capture ly in low ionic strength (<10 mM) EDTA-containing buffers. A few reports have examined the properties of PNAs as both specific [7,26,27] as well as general [28] nucleic acid Considering the range of properties of PNAs, it is not surpris- capture probes (for example, for sample preparation), tak- ing that the most successful applications of PNAs are those ing advantage, in particular, of the tight complex formation that purposely or serendipitously take advantage of and at low ionic strength under which nucleic acid secondary exploit the special properties of PNAs that distinguish them structure is destabilised significantly [7]. Two recent from oligonucleotides (see [9–12] for recent reviews on PNAs reports [26,27] analysed in more detail the performance of and their applications). In this review the recent advances in PNA oligomers compared with DNA oligomers for capture using PNA and developing PNA-based technologies for and recovery of 16S ribosomal DNA from very dilute genetic diagnostics and molecular biology are discussed. (i.e. femtomolar) samples. The results indicated no gener- al advantage to using PNAs, but also (not surprisingly) In situ hybridisation showed that both the absolute and relative performance of PNA probes have proven extremely useful for use in a vari- PNA and DNA capture probes is heavily dependent on the ety of FISH (fluorescence in situ hybridisation) assays, experimental conditions (e.g. ionic strength). These 18 Analytical biotechnology

Figure 3 Figure 4

(a) B

NH N

DNA oligo PNA PNA O Current Opinion in Biotechnology

Chemical structure of aep–PNA (N-[2-aminoethyl]propyl–PNA).

(b) designed [31•] (Figure 3b,c). Analogous to the pad-lock hybridisation system [32], this principle could be devel- oped for signal-amplified in situ hybridisation techniques.

Ligation vector tagging The extremely stable and highly sequence-specific com- plexes formed upon triplex invasive binding of homopyrimidine PNAs to dsDNA has been exploited as a means of tagging plasmid DNA vectors with fluorophores [33], targeting [34] and, most recently, with a vari- (c) ety of ligands through a biotin–streptavidin ‘sandwich’ linker [35•]. Essentially, this technology allows an almost irreversible, yet noncovalent and therefore almost ‘biolog- ically silent’, labelling of DNA molecules that can be used Earring complex for transfection and eventually for . Current Opinion in Biotechnology Duplex DNA targeting Schematic representation of PD loop. (a) Two homopyrimidine ‘PNA A major step towards general sequence targeting of openers’ binding to closely positioned sites create one large DNA loop dsDNA by PNA, as opposed to the homopurine restric- to which an can bind. Such a complex may be used to capture the DNA (e.g. via streptavidin beads) by employing a tions inherent in the triplex invasion binding mode, was biotinylated oligonucleotide. (b) The complex may be detected by achieved by introducing pseudocomplementary PNAs that DNA-polymerase-mediated extension of the oligonucleotide with bind to duplex DNA targets via the double duplex inva- radiolabelled precursors. (c) Finally, the complex may be used for sion mode [8••] (Figure 2). Most, if not all, of the template-directed ligation (cyclisation) of oligonucleotide, which may result in an ‘earring’ complex. techniques developed based on PNA triplex invasion could take advantage of this new binding mode. Moreover, recently it was demonstrated that a rare cleavage tech- results again stress that optimisation of PNA- and nique exploiting inhibition of specific DNA methylation DNA-based techniques are different. sites and subsequent restriction cleavage of these sites could readily be extended to sites protected by A rather complex, but nonetheless ingenious, system for pseudocomplementary PNAs [36]. capture of dsDNA was devised recently which employed a PD loop complex [29]. This complex is composed of two Detection by solution-phase hybridisation PNA (triplex invasion) openers and an oligonucleotide Direct, real-time detection of DNA targets, for example dur- hybridizing to the single-stranded loop in between these ing a polymerase chain reaction (PCR), is possible using (Figure 3a). This system relies on simultaneous sequence- DNA oligonucleotides through beacon technology [37]. specific binding of three ligands (two PNAs and one The beacon technology takes advantage of a partly self- oligonucleotide) and therefore shows exquisite sequence complementary oligo() specificity, allowing capture of single chromosomes (so far containing a fluorophore at one end and a quencher at the only from yeast). PD loops also may be used for direct other end. When the probe is not hybridized to the target, it detection of specific DNA sequences, for example, in intact forms a hairpin that does not fluoresce because of the close chromosomes, by simple enzymatic primer extension of the proximity of the quencher and the fluorophore; however, bound oligonucleotide with radiolabelled upon hybridizaton, the hairpin is opened, the quencher is triphosphates [30••]. As an extension of this principle, a moved away from the fluorophore and the probe fluoresces. locked earring complex, in which a single-stranded, circular PNA beacons also have been developed [38], but it is as yet oligonucleotide is interlocked into the DNA helix, was unclear if they have any advantages over oligonucleotide Peptide nucleic acid: a versatile tool in genetic diagnostics and molecular biology Nielsen 19

(natural or analogues) beacons. It may be possible, however, Figure 4) was shown — at least in certain sequence con- to exploit the charge-neutral, non-DNA properties of PNAs texts — to provide very significant stabilisation. If this in order to develop PNA–fluorophore conjugates that stabilisation is a general property of PNAs containing aep specifically fluoresce only upon binding to a DNA (or RNA) backbone modifications, aep–PNAs could offer great target. Some dyes, such as thiazole orange (TO), exhibit a advantages in many of the applications discussed above. three orders of magnitude increase in fluorescence quantum yield upon binding to both single-stranded or dsDNA, but References and recommended reading does so in an analogous manner to other DNA-binding Papers of particular interest, published within the annual period of review, agents [39] (i.e. they do not bind efficiently to PNA). have been highlighted as: Accordingly, it was shown recently that some TO–PNA con- • of special interest •• of outstanding interest jugates can be used as hybridisation ‘light-up’ probes [40••]. 1. Nielsen PE, Egholm M, Berg RH, Buchardt O: Sequence-selective Unfortunately, this effect appears to be very dependent on recognition of DNA by strand displacement with a thymine- the PNA sequence and the mechanisms underlying this substituted polyamide. Science 1991, 254:1497-1500. dependence are as yet not fully understood. 2. Nielsen PE, Christensen L: Strand displacement binding of a duplex-forming homopurine PNA to a homopyrimidine duplex The charge-neutral nature of PNAs also has been exploit- DNA target. J Am Chem Soc 1996, 118:2287-2288. ed in an ‘in-gel affinity’ separation technique for 3. Giesen U, Kleider W, Berding C, Geiger A, Ørum H, Nielsen PE: A formula for thermal stability (Tm) prediction of PNA/DNA separating long DNA molecules that only differ by a single duplexes. Nucleic Acids Res 1998, 26:5004-5006. nucleobase. This was accomplished simply by inclusion of 4. SantaLucia J, Allawi H T, Seneviratne PA: Improved nearest- a complementary PNA in the polyacrylamide gel during neighbor parameters for predicting DNA duplex stability electrophoresis [41•]. Biochemistry 1996, 35:3555-3562. 5. Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, PCR clamping Driver DA, Berg RH, Kim SK, Norden B, Nielsen PE: PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick Inhibition of PCR amplification of a specific target by hydrogen-bonding rules. Nature 1993, 365:566-568. ‘PCR clamping’ [42] (by which a PNA oligomer is used to 6. Tomac S, Sarkar M, Ratilainen T, Wittung P, Nielsen PE, Norden B inhibit the amplification of a specific target, e.g., by direct et al.: Ionic effects on the stability and conformation of peptide competition with a PCR primer) has been used very suc- nucleic acid complexes. J Am Chem Soc 1996, 118:5544-5552. cessfully to detect and screen for single base-pair gene 7. Ørum H, Nielsen PE, Jørgensen M, Larsson C, Stanley C, Koch T: Sequence-specific purification of nucleic acids by PNA-controlled variants [43,44]. The technique is so powerful, in fact, that hybrid selection. BioTechniques 1995, 19:472-480. it also can be used to detect single base-pair gene variants • 8. Lohse J, Dahl O, Nielsen PE: Double duplex invasion by peptide on a background of up to a 100-fold excess wild type [45 ]. •• nucleic acid: a general principle for sequence-specific targeting of This is accomplished most efficiently by suppressing double-stranded DNA. Proc Natl Acad Sci USA 1999, 96:11804-11808. amplification of the wild-type allele with a PNA that is The double duplex invasion mechanism for targeting DNA with PNA complementary to the PCR-primer-binding site of the wild described in this paper in principle allows targeting of any sequence in duplex type allele, thereby effectively competing with primer DNA, but the technology is so far limited to sequences containing at least 50% AT bases. The technique is used in [36] in the ‘Achilles Heel’ approach. binding to this allele but much less efficiently with binding 9. Nielsen PE, Egholm M (eds): Peptide Nucleic Acids: Protocols and of the primer for the variant gene. Applications. Wymondham, UK: Horizon Scientific Press;1999. 10. Ganesh KN, Nielsen PE: Peptide nucleic acids. Analogs and Similarly, preferential cloning of gene variants can exploit derivates. Curr Org Chem 2000, 4:916-928. the PCR clamping technique for gene isolation [46•] or 11. Nielsen PE: Antisense peptide nucleic acids. Curr Opin Mol Ther genetic analyses [47]. Finally, studies of mRNA editing can 2000, 2:282-287. take advantage of the PNA PCR clamping technique by 12. Ray A, Norden B: Peptide nucleic acid (PNA): its medical and suppressing the major signal from the non-edited form [48]. biotechnical applications and promise for the future. FASEB J 2000, 14:1041-1060. Conclusions and future perspectives 13. Lansdorp PM, Verwoerd NP, Van de Rijke FM, Dragowska V, Little M-T, Dirks RW, Raap AK, Tanke HJ: Heterogeneity in telomere length of In the past year, it has become clear that the use of PNAs human chromosomes. Hum Mol Genet 1996, 5:685-691. in in situ hybridisation and PCR clamping are becoming 14. Ulhmann V, Prasad M, Silva I, Luettich K, Grande L, Alonso L, established techniques that have wide and expanding Thisted M, Pluzek KJ, Gorst J, Ring M et al.: Improved in situ applications. New applications of PNAs also are still detection method for telomeric tandem repeats in metaphase emerging, and there is no reason to believe that this trend spreads and interphase nuclei. Mol Pathol 2000, 53:48-50. shall not continue. Many novel chemical modifications of 15. Surrallés J, Hande MP, Marcos R, Lansdorp PM: Accelerated telomere shortening in the human inactive X chromosome. Am J the original aminoethyl glycine PNA backbone have been Hum Genet 1999, 65:1617-1622. made and are still being developed [10]. Most of these are 16. Kajstura J, Pertoldi B, Leri A, Beltrami CA, Deptala A, Darzynkiewicz Z, not interesting in relation to practical applications, but Anversa P: Telomere shortening is an in vivo marker of myocyte some increase the stability of PNA–DNA/RNA hybrids. replication and aging. Am J Pathol 2000, 156:813-819. Novel, conformationally restricted (i.e. cyclic) PNA back- 17. Deng W, Lucas JN: Combined fish with pan-telomeric PNA and bones show promise in this direction in particular; and whole chromosome-specific DNA probes to detect complete and incomplete chromosomal exchanges in human lymphocytes. Int J recently, aep–PNA (N-[2-aminoethyl]propyl–PNA; Radiat Biol 1999, 75:1107-1112. 20 Analytical biotechnology

18. Fomina J, Darroudi F, Boei JJWA, Natarajan AT: Discrimination 33. Zelphati O, Liang X, Hobart P, Felgner PL: Gene chemistry: between complete and incomplete chromosome exchanges in functionally and conformationally intact fluorescent plasmid. X-irradiated human lymphocytes using FISH with pan-centromeric Hum Gene Ther 1999, 10:15-24. and chromosome specific DNA probes in combination with telomeric PNA probe. Int J Radiat Biol 2000, 76:807-813. 34. Branden LJ, Mohamed AJ, Smith CIE: A peptide nucleic acid- nuclear localization signal fusion that mediates nuclear transport 19. Boei JJWA, Vermeulen S, Natarajan AT: Analysis of radiation- of DNA. Nat Biotechnol 1999, 17:784-787. induced chromosomal aberrations using telomeric and centromeric PNA probes. Int J Radiat Biol 2000, 76:163-167. 35. Zelphati O, Liang X, Nguyen C, Barlow S, Sheng S, Shao Z, • Felgner PL: PNA-dependent gene chemistry: stable coupling of 20. Chen C, Wu BL, Wei T, Egholm M, Strauss WM: Unique peptides and oligonucleotides to plasmid DNA. BioTechniques • chromosome identification and sequence-specific structural analysis 2000, 28:304-316. with short PNA oligomers. Mamm Genome 2000, 11:384-391. Tagging of vector DNA via PNA triplex invasion complex formation is pre- This paper reports a series of chromosome specific PNA in situ hybridisa- sented. This technology should have wide applications in gene therapy. tion probes [9] that can be used to identify chromosomes. The aim is to iden- tify specific PNAs for all human chromosomes. 36. Izvolsky KI, Demidov VV, Nielsen PE, Frank-Kamenetskii MD: Sequence-specific protection of duplex DNA against restriction 21. Drobniewski FA, More PG, Harris GS: Differentiation of Mycobacterium and methylation by pseudocomplementary PNAs. tuberculosis complex and nontuberculous mycobacterial liquid Biochemistry 2000, 39:10908-10913. cultures by using peptide nucleic acid-fluorescence probes. J Clin Microbiol 2000, 38:444-447. 37. Piatek AS, Tyagi S, Pol AC, Telenti A, Miller LP, Kramer FR, Alland D: sequence analysis for detecting drug resistance 22. Stender H, Mollerup TA, Lund K, Petersen KH, Hongmanee P, in Mycobacterium tuberculosis. Nat Biotechnol 1998, 16:359-363. Godtfredsen SE: Direct detection and identification of Mycobacterium tuberculosis in smear-positive sputum samples 38. Seitz O: Solid-phase synthesis of doubly labeled peptide nucleic by fluorescence in situ hybridization (FISH) using peptide nucleic acids as probes for the real-time detection of hybridization. acid (PNA) probes. Int J Tuberc Lung Dis 1999, 3:830-837. Angew Chem Int Ed 2000, 39:3249-3252. 23. Stender H, Lund K, Petersen KH, Rasmussen OF, Hongmanee P, 39. Wittung P, Kim SK, Buchardt O, Nielsen P, Norden B: Interactions of Miorner H, Godtfresden SE: Fluorescence in situ hybridization DNA binding ligands with PNA-DNA hybrids. Nucleic Acids Res assay using peptide nucleic acid probes for differentiation 1994, 22:5371-5377. between tuberculous and nontuberculous Mycobacterium species 40. Svanvik N, Westman G, Wang D, Kubista M: Light-up probes: in smears of Mycobacterium cultures. J Clin Microbiol 1999, •• thiazole orange-conjugated peptide nucleic acid for detection of 37:2760-2765. target nucleic acid in homogeneous solution. Anal Biochem 2000, 24. Padilla E, Manterola JM, Rasmussen OF, Lonca J, Dominguez J, 281:26-35. • Matas L, Hernandez A, Ansina V: Evaluation of a fluorescence A previous observation [39] that DNA-binding ligands in general do not bind hybridisation assay using peptide nucleic acid probes for the charge-neutral PNA is exploited in this study to design PNA-thiazole identification and differentiation of tuberculous and non- orange conjugates that only fluoresce upon binding to a complementary tuberculous mycobacteria in liquid cultures. Eur J Clin Microbiol DNA (or RNA) target. This idea was realised to a certain degree, but unfor- Infect Dis 2000, 19:140-145. tunately many PNA-thiazole orange conjugates (depending on the PNA One of the first ‘clinical evaluations’ of the use of a PNA in situ hybridisation sequence) fluoresce intensely, and no fluoresence increase occurs upon assay for diagnosis of tuberculosis. hybridisation. Thus, although this very attractive principle is valid, the probe sequences have to be carefully and, so far, empirically selected. 25. Prescott AM, Fricker CR: Use of PNA oligonucleotides for the • in situ detection of Escherichia coli in water. Mol Probes 41. Igloi GL: Automated detection of point mutations by 1999, 13:261-268. • electrophoresis in peptide-nucleic acid-containing gels. This paper describes an interesting new application within environmental BioTechniques 1999, 27:798-808. monitoring for PNA in situ hybridisation. This method could gain wide use An ingenious way of exploiting the charge-neutral nature of PNA for in gel analy- for testing and screening of bacterial contamination of water samples. sis and separation of larger DNA fragments only differing by point mutations. 26. Chandler DP, Stults JR, Anderson KK, Cebula S, Schuck BL, 42. Ørum H, Nielsen PE, Egholm M, Berg RH, Buchardt O, Stanley C: Brockman FJ: Affinity capture and recovery of DNA at femtomolar Single mutation analysis by PNA directed PCR concentrations with peptide nucleic acid probes. Anal Biochem clamping. Nucleic Acids Res 1993, 21:5332-5336. 2000, 283:241-249. 43. Behn M, Schuermann M: Sensitive detection of p53 gene 27. Chandler DP, Stults JR, Cebula S, Schuck BL, Weaver DW, mutations by a ‘mutant enriched’ PCR–SSCP technique. Nucleic Anderson KK, Egholm M, Brockman FJ: Affinity purification of DNA Acids Res 1998, 26:1356-1358. and RNA from environmental samples with peptide nucleic acid clamps. Appl Environ Microbiol 2000, 66:3438-3445. 44. Murdock DG, Christacos NC, Wallace DC: The age-related accumulation of a mitochondrial DNA control region mutation in 28. Seeger C, Batz H-G, Ørum H: PNA-mediated purification of PCR muscle, but not brain, detected by a sensitive PNA-directed PCR amplifiable human genomic DNA from whole blood. BioTechniques clamping based method. Nucleic Acids Res 2000, 28:4350-4355. 1997, 23:512-516. 45. Behn M, Thiede C, Neubauer A, Pankow W, Schuermann M: 29. Bukanov NO, Demidov VV, Nielsen PE, Frank-Kamenetskii MD: • Facilitated detection of oncogene mutations from exfoliated PD-loop: a complex of duplex DNA with an oligonucleotide. tissue material by a PNA-mediated ‘enriched PCR’ protocol. Proc Natl Acad Sci USA 1998, 95:5516-5520. J Pathol 2000, 190:69-75. A very illustrative and convincing example of the versatility of the PCR clamp- 30. Broude NE, Demidov VV, Kuhn H, Gorenstein J, Pulyaeva H, ing technique for detecting and analysing low-abundance oncogene muta- •• Volkovitsky P, Drukier AK, Frank-Kamenetskii MD: PNA openers as tions in clinical material. a tool for direct quantification of specific targets in duplex DNA. J Biomol Struct Dyn 1999, 17:237-244. 46. Cochet O, Martin E, Fridman WH, Teillaud J-L: Selective PCR The PD loop complex is exploited to initiate primer extension with extreme • amplification of functional immunoglobulin light chain from specificity in a double-stranded DNA target. This technology may be devel- hybridoma containing the aberrant MOPC 21-derived Vκ by oped into a very sensitive and specific method for quantification of DNA tar- PNA-mediated PCR clamping. BioTechniques 1999, 26:818-822. gets in various biological and medical samples. The first example showing the power of the PCR clamping technique for enrichment of the gene of interest for cloning. 31. Kuhn H, Demidov VV, Frank-Kamenetskii MD: Topological links • between duplex DNA and a circular DNA single strand. Angew 47. Von Wintzingerode F, Landt O, Ehrlich A, Gobel UB: Peptide nucleic Chem Int Ed 1999, 38:1446-1449. acid-mediated PCR clamping as a useful supplement in the The ‘earring’ DNA complex described in this paper may be exploited for sig- determination of microbial diversity. Appl Environ Microbiol 2000, nal amplification by rolling circle replication as described in [32]. 66:549-557. 32. Baner J, Nilsson M, Mendel-Hartvig M, Landegren U: Signal 48. Zhong S, Nguyen NY, Eggerman TL: Detection of apolipoprotein B amplification of padlock probes by rolling circle replication. mRNA editing by peptide nucleic acid mediated PCR clamping. Nucleic Acids Res 1998, 26:5073-5078. Biochem Biophys Res Commun 1999, 259:311-313.