SNuPE - Biochemical Society Transactions

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The single-nucleotide primer extension (SNuPE) method for the multiplex detection of various DNA sequences: from detection of point mutations to microbial ecology ´ Tancsics‡, ´ ¨ Imfeld§ and Matthias Kastner* ¨ Marcell Nikolausz*1 , Antonis Chatzinotas†, Andras Gwenael *Helmholtz Centre for Environmental Research, UFZ, Department of Environmental Biotechnology, Permoserstrasse 15, D-04318 Leipzig, Germany, †Helmholtz Centre for Environmental Research, UFZ, Department of Environmental Microbiology, Permoserstrasse 15, D-04318 Leipzig, Germany, ‡Department of Microbiology, Eotv ¨ os ¨ Lorand ´ University of Science, Pazm ´ any ´ Peter ´ set ´ any ´ 1/c, 1117 Budapest, Hungary, and §Helmholtz Centre for Environmental Research, UFZ, Department of Isotope Biogeochemistry, Permoserstrasse 15, D-04318 Leipzig, Germany

Abstract Methods based on SNuPE (single-nucleotide primer extension) have become invaluable tools for the rapid and highly specific detection of point mutations and single-nucleotide polymorphisms in the field of human genetics. In the primer extension reaction, a DNA polymerase is used to label a specific primer hybridized to the target sequence by incorporating a single labelled ddNTP (dideoxynucleotide). This labelling provides not only information about the complementary nucleotide of interest in the opposite strand but also a semiquantitative analysis of the sequence targeted by the primer. Since several subdisciplines of microbiology increasingly require cultivation-independent molecular screening tools for elucidating differences between either strains or community structures based on sequence variations of marker genes, SNuPE offers a promising alternative to the existing tool box. The present review describes the method in detail and reports the state-of-the-art applications of this technique both in the field of nucleic acid detections in human genetics and in microbiology.

Introduction A major driving force for the development of novel molecular biological diagnostic tools is to find easier and faster ways for the determination of point mutations causing genetic disorders and analysing SNPs (single-nucleotide polymorphisms) responsible for phenotypic variations. Recently, these techniques have gained popularity in scientific disciplines other than human genetics. Several methods originally developed for screening point mutations became valuable molecular detection or ‘fingerprinting’ tools for the fast and easy assessment of microbial community structures in environmental samples. Methods based on SNuPE (singlenucleotide primer extension) hold this potential for several applications in microbiology. This approach was developed for the analysis of single-nucleotide positions of various genes and, because of its simplicity, it became very popular in the analysis of hereditary diseases, tissue typing for histocompatibility, forensic analysis, parental testing, prenatal diagnostics and sequence typing of different organisms. The present review summarizes the technical aspects of the most commonly used techniques relying on primer extension and, beyond Key words: microbiology, minisequencing, multiplex screening, mutation detection, singlenucleotide polymorphism, single-nucleotide primer extension (SNuPE). Abbreviations used: ddNTP, dideoxynucleotide; FISH, fluorescence in situ hybridization; FNC, first nucleotide change; SNP, single-nucleotide polymorphism; SNuPE, single-nucleotide primer extension. 1 To whom correspondence should be addressed (email [email protected]).

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mutation analysis, evaluates their potential applications with particular emphasis on microbiological aspects. The principle of SNuPE is described in Figure 1. The method benefits from the high fidelity of DNA polymerases while incorporating nucleotides or nucleotide analogues, resulting in a highly specific distinction of sequence variants. When a specific primer hybridizes upstream from the target nucleotide position, a DNA polymerase incorporates a labelled nucleoside triphosphate, which terminates the reaction and results in a labelled extended primer. This labelling provides information about the nucleotide of interest in the opposite strand. The high accuracy of this incorporation is due to (i) preferential binding of the dNTP substrate to the enzyme–DNA complex, (ii) faster phosphodiester bond formation of the correct enzyme–DNA–dNTP complex, and (iii) a more rapid rate of PPi release in the case of correct dNTP incorporation [1]. In addition, the proofreading activity of a DNA polymerase contributes to the fidelity of the reaction. However, recent primer extension assays mainly use 3 → 5 exonuclease-deficient (non-proofreading) DNA polymerases. Sokolov [2] demonstrated the applicability of primer extension for the determination of a single nucleotide in genomic DNA. In this proof of principle study, four parallel cycle reactions were performed using one radioisotopelabelled nucleotide of the four followed by the separation of the products by gel electrophoresis. The first application Biochem. Soc. Trans. (2009) 37, 454–459; doi:10.1042/BST0370454

Advances in Nucleic Acid Detection and Quantification

Figure 1 The principle of the SNuPE method A detection primer anneals upstream from the nucleotide of interest. A modified Taq DNA polymerase incorporates a labelled ddNTP, which terminates the reaction and results in a labelled product. The type and amount of labelling provide information about a single base in the opposite strand and about the hybridization efficiency.

focused on genotyping apolipoprotein E using biotinlabelled PCR products as a template [3]. After linearization of the PCR product and capturing on an avidin matrix, a single-step reaction incorporated the radioisotope-labelled ddNTPs (dideoxynucleotides). These premiere studies have been followed by a wide range of applications differing mainly either in the choice of labelling, the separation method or the way of multiplexing. Radioisotope labelling has been gradually replaced by fluorescent techniques, and size separation by capillary electrophoresis has gained increased popularity. Although SNuPE [4] and ‘minisequencing’ [5] are the most widely used terms for these assays, the same principle has been applied under different names, such as SBE (single-base extension) [6], TDI (template-directed dye-terminator incorporation) [7], FNC (first nucleotide change) [8] and PROBE (primer oligo base extension) [9], just to mention the most popular ones.

Labelling and separation The pioneer primer extension assays applied nucleoside triphosphates labelled with 32 P, 33 P or 3 H to detect the extension event [4,10,11]. The labelled primers were separated from the templates by gel electrophoresis, and autoradiography was used for visualization and quantification of the signal. The size separation enabled the application of several primers differing in size per reaction and hence targeting more than one SNP [10]. Another format used solid supports for immobilizing one of the reactants usually via biotin–avidin interactions. Biotin is introduced typically by one of the primers used for generating the target PCR product, which is then captured in microtitre plate wells [5,12] or with magnetic microparticles [13,14] covered by avidin. Denaturation of the PCR amplicons generates single-stranded DNA for more effective primer–template hybridization, whereas the

remaining PCR reactants that may interfere with the primer extension reactions can be washed away with the nonbound strand. Using double-stranded DNA as a template may require cyclic primer extension reactions. Alternatively, the detection primers were attached to a solid support covalently [15,16] or via biotin–avidin interactions [8] before the reaction. Haptenes were introduced as alternatives to radioisotopes for labelling ddNTPs, allowing colorimetric or chemiluminescent detection assays catalysed by alkaline phosphatase or peroxidase conjugated with the haptenebinding antibody [3,17,18]. Although these assays were accurate and very sensitive, the possibility of multiplex detection is precluded owing to the homogeneous labelling. Applying ddNTPs that are each labelled with different fluorescent dyes enables the detection of the base variations in one single reaction. In addition, using different target-specific primers also varying in length allows the analysis of more than one locus (up to 8–12) in one reaction. In the first studies, automated DNA sequencers equipped with four-fluorophore detection systems were used for size separation of the extended products and for the simultaneous detection of the incorporated fluorophore via laser-induced fluorescence [19,20]. Combined with the higher resolution power of capillary electrophoresis as compared with that of gel-based systems, a more accurate size separation of the extended products could be achieved [21–24]. A similar multiplex version of SNuPE uses MALDI–TOFMS (matrix-assisted laser-desorption ionization–time-offlight MS) [25]. Owing to the small mass difference between the various nucleotides, chemically modified ddNTPs with molecular tags are used to increase the mass difference and to improve the separation of the differently labelled products [26]. HPLC [27] or DHPLC (denaturing HPLC) can also be used to separate the extended primers [28,29].  C The

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The main limitation of the size separation is that the primers have to bear a non-complementary mobility modifier tail, which influences the hybridization and hence the primer extension efficiency. This limitation can be circumvented by adding a unique oligonucleotide ‘tag’ to the 5 -end of the detection primers. The extension product can then be sorted by hybridizing the ‘tags’ to complementary ‘antitags’ attached to an array or bead (microsphere) surface. Depending on the type of labelling of the array, fluorescent scanning [30,31] or phosphoimager instruments might be used for subsequent analysis [16,32]. Tagged extended primers can also be captured on microspheres having distinct spectral properties and carrying complementary antitags. Flow cytometric analysis allows the separation of different microspheres and the typing of the captured extension products [33,34]. The last decade saw a bloom of different assays applying the principle of primer extension both in basic research and routine diagnostics [18,23,35–37]. Readers who are interested in the application of primer extension in human genetics in more detail should consult the more comprehensive reviews [38–40].

Microbiological application of SNuPE Although assays based on primer extension have been mainly used in the field of human genetics, the approach has recently found its way to other disciplines. One of the main aspects of modern microbiology is the precise taxonomic affiliation of closely related bacterial strains. Primer extension based on the characterization of multiple nucleotide positions with taxonomical value thus provides a promising alternative to MLST (multilocus sequence typing) [41] in microbiological diagnostics. Surprisingly, only a limited number of primer extension-based characterizations of bacteria have been reported, including the phylotyping of Listeria monocytogenes [42,43] and Escherichia coli strains [44] and the rapid identification of Brucella isolates [45]. These studies demonstrated the good discrimination potential and high taxonomical resolution of SNP analyses with primer extension. A similar approach can be used for a wide variety of micro-organisms as soon as a representative reference collection is available and a sequence database is established. Such a typing approach can be completed in 1 day, which is a clear time and cost benefit compared with sequencing several DNA fragments or performing classical biotyping assays. Microbial ecology increasingly relies on cultivationindependent molecular methods for monitoring complex microbial communities in ecosystems and microbial source tracking. PCR-based approaches are believed to overcome some of the biases inherent to cultivation and provide reliable and rapid information. These approaches commonly target phylogenetic marker genes, such as the 16S or 18S rRNA gene. Several attributes of the SNuPE make it an ideal molecular detection tool for microbial ecology: the primer extension technique not only allows detection of point mutations but can also be used to detect a hybridization event and hence the detection of longer sequence stretches complementary to the selected primers. Compared with RNA or DNA probe  C The

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technologies, i.e. FISH (fluorescence in situ hybridization), a high discrimination potential is ensured not only by a matching primer–template structure, but also by the selective ability of the DNA polymerase to continue DNA synthesis only with a matching 3 -end. Consequently, the discriminating power of primer extension can be achieved by taking into account both terminal and internal mismatch positions. Ideally, signature sequences unique to the target micro-organisms can be used for designing detection primers. Subsequently, hybridized detection primers will gain a fluorescent label via incorporation of the complementary fluorescent nucleotide in the SNuPE reaction. The nucleotide may provide additional taxonomic information and the fluorescence signal can be used in a semiquantitative way once the correlation between signal intensity and template abundance has been validated and established. Rudi et al. [46] developed the first primer extension-based application for the detection of toxic cyanobacteria by labelling only one incorporated ddNTP from the four used. Multiplexing was achieved by hybridization of the labelled products to complementary oligonucleotides on an array format, which is similar to the ‘tag–antitag’ array design without the possibility of reusing the array for other targets. An antibody-based chromogenic detection was used for visualization of the results. This approach was applied for profiling of cyanobacteria diversity from lake samples [47] and for describing microbial communities in ready-to-eat vegetable salads in a modified atmosphere [48]. However, homogeneous labelling of only one of the four ddNTPs restricts the primer design to certain sequence positions, and the type of the incorporated terminator nucleotide cannot provide additional taxonomical information. In order to exploit the above outlined potential of SNuPE, we recently combined ddNTPs that are each labelled with different fluorescent dyes and different target-specific primers varying in length in one reaction. The extended products were separated by capillary electrophoresis, and the type and amount of labelling were measured by laser-induced fluorescence detection. Our first multiplex SNuPE assay was developed for the detection and typing of ‘Dehalococcoides’ spp. sequences obtained from chloroethene-contaminated groundwater samples [24]. Primer design was aimed at discriminating three major phylogenetic subgroups of the genus Dehalococcoides (i.e. Cornell, Victoria and Pinellas) and at demonstrating the potential of SNuPE as a detection and typing method for environmental applications. Two primers had two mismatches against the DNA template from the non-target Cornell subgroup, which was sufficient to prevent primer extension. Further discrimination of sequence types was achieved by the typing of the incorporated single nucleotide. In this way, the multicolour detection system provided more flexibility than previous minisequencingbased detection methods using only one labelled ddNTP. The predicted taxon-specific pattern was verified with positive control templates and template mixtures, and 16S rRNA gene PCR products obtained from contaminated groundwater samples could successfully be screened by the

Advances in Nucleic Acid Detection and Quantification

assay. Moreover, a linear relationship was observed between the detected fluorescent signal and the applied template concentration, which underscored some quantitative potential of the approach [24]. An improved version of SNuPE was developed by Wu and Liu [49], including detection primers at different taxonomical levels. Primers complementary to more conservative regions of the rRNA gene sequence representing higher taxonomical levels were used as an internal standard, whereas lower hierarchy level primers can be calibrated to them by comparing their extension efficiency. The method, termed HOPE (hierarchical oligonucleotide primer extension) [49], was successfully applied for the determination of relative abundance of predominant Bacteroides spp. present in wastewater and fecal samples [50]. The results showed good agreement with those obtained with quantitative PCR of the same samples and previous results based on FISH, cloning and sequencing of the 16S rRNA genes. Similar to most molecular biological techniques, the specificity and sensitivity of the SNuPE is of paramount importance. Although systematic studies on the factors affecting primer design and discrimination power of the primer extension are still lacking, only a few reports deal with these crucial issues (M. Nikolausz, A. Chatzinotas, A. T´ancsics, G. Imfeld and M. K¨astner, unpublished work). Using primers with introduced single and multiple mismatches confirmed that a 3 terminal mismatch has the most significant effect on primer extension probably because of the fidelity of the extension reaction, whereas mismatches in the middle of the primer destabilize the hybrid duplex. Consequently, carefully chosen primer-mismatch positions result in a high signal-to-noise ratio and prevent any non-specific extension (M. Nikolausz, A. Chatzinotas, A. T´ancsics, G. Imfeld and M. K¨astner, unpublished work). The detection limit was approx. 106 –107 copies of template per μl of primer extension reaction mixture, which indicates that a preamplification step is necessary. However, the development of more sensitive detection sensors along with the improvement and discovery of new fluorophores will probably further enhance this sensitivity. Since quantitative biases are in general associated with PCR-based approaches when applied to complex microbial communities [51,52], reverse-transcribed rRNA might be considered as a potential naturally amplified (i.e. abundant) target molecule. We successfully demonstrated the applicability of reverse-transcribed RNA as a template without further pre-amplification steps (M. Nikolausz, A. Chatzinotas, A. T´ancsics, G. Imfeld and M. K¨astner, unpublished work). MDA (multiple displacement amplification), using φ29 DNA polymerase and random primers in an isothermal reaction [53], is another option as a potential preamplification step which should be investigated in the future. Although the capillary electrophoresis-based separation limits the number of detection primers per reaction, several parallel reactions with additional primers are easy to set up, including a common primer that serves as an internal standard to achieve tube-to-tube comparison [49]. Another way of increasing multiplexing capability is to apply the high-

density tag-array format in microbiological experiments. In conclusion, keeping the PCR biases in mind and the challenge of designing rapid quantitative diagnostic tools, SNuPE is a very promising method for the simultaneous and (at least) semiquantitative detection of multiple microbial target organisms in environmental or engineered samples.

Future perspectives Diagnostic methods based on the principle of SNuPE have been proved to be very rapid, sensitive and robust; furthermore, scaling up from medium to very high throughput applications seems feasible. The versatility and simplicity of this method can further facilitate its successful application in different fields of microbiology. Several advantages of the method were already demonstrated in the few early studies in microbiology, but the full potential of this method has not been exploited yet. We believe that SNuPE is an invaluable tool for the rapid, reliable and simultaneous detection of several signature sequences representing different micro-organisms. For instance, the application of various primer-extension methods could be successfully expanded to clinical and food microbiology due to its capacity for very rapid cultivation-independent detection, which is required for effective and low-cost pathogen control programmes. In addition, SNuPE can be established as an invaluable tool in environmental microbiology to follow spatial and temporal changes in the relative abundance of target micro-organisms in various environmental samples. However, confrontation with the huge unknown diversity in environmental systems requires the development of precise databases and novel bioinformatics tools for the primer design. On the other hand, the ability of exploiting an SNP for detection and tracking purposes gives SNuPE an advantage over other molecular ‘fingerprinting’ techniques while providing excellent taxonomic resolution.

Funding The project was supported financially by the Helmholtz Centre for Environmental Research, UFZ. G. I. was supported by the Marie Curie Early Stage Training project of the European Union [AXIOM contract number MEST-CT-2004-8332]. We thank Sachsisches ¨ Staatsministerium fur ¨ Wissenschaft and Kunst for providing a grant [contract number 4-7531.50-04-840-07/3] for A. T.

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Received 26 October 2008 doi:10.1042/BST0370454

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