Journal of Analytical & Molecular Techniques
Single Nucleotide Polymorphism Detection Using a Biocompatible, Fluorogenic and DNA-Templated Reaction of Cyanine Dye Formation
Kamel Meguellati1, Girish Koripelly1 and Sylvain Ladame1,2*
- 1ISIS Universite de Strasbourg, 8 allée Gaspard Monge, BP 70028, 67083 Strasbourg, France
- 2Department of Bioengineering, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K
*Address for Correspondence: Sylvain Ladame, Department of Bioengineering, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K, Tel: (+44) 02075945308; E-mail: firstname.lastname@example.org
Citation: Meguellati K, Koripelly G, Ladame S. Single Nucleotide Polymorphism Detection Using a Biocompatible, Fluorogenic and DNA-Templated Reaction of Cyanine Dye Formation. J Analyt Molecul Tech 2013;1(1): 5.
Copyright © 2013 Ladame et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Journal of Analytical & Molecular Techniques |ISSN: 2474-1914 | Volume: 1, Issue: 1
Submission: 19 September 2013 | Accepted: 13 December 2013 | Published: 18 December 2013
AbstractWater-soluble Peptide Nucleic Acids (PNAs) functionalised with fluorogenic cyanine dye precursors (i.e. a Fisher’s base aldehyde and a 2-methylene indolenine) are employed for detecting single-nucleotide polymorphism in vitro under near physiological conditions of salt and pH. This quick and versatile fluorogenic system has been designed so that fluorescence is formed only upon simultaneous hybridisation of both PNAs to a fully complementary DNA strand. The reaction yield increased up to around 10% upon addition of the DNA template, even in the presence of a large excess of Calf-Thymus competitor DNA.
IntroductionSince the final sequencing of the human genome was completed, in 2003, considerable research effort has been devoted to the identification and analysis of variations among individual genomes [1-4]. Although more than 99.9% of human DNA sequences are the same across the population, variations can have a major impact on how humans respond to diseases or drug therapies [5,6]. Amongst the most common forms of genomic variation, single-nucleotide polymorphism (SNP) accounts for over 80% of sequence variants [7-9]. Current estimates suggest that SNPs occur as frequently as every 100 to 300 bases and that they are stably inherited, making them a unique tool/marker for identifying numerous genetic and inherited complex diseases such as cancers and diabetes [7-9]. Therefore, there is an increasing need for high throughput technologies suitable for rapid and sensitive SNP genotyping [10-12]. So far, methods using short oligonucleotides or oligonucleotide mimics as probes to reveal the presence of complementary sequences have been successfully developed [13,14]. Of particular interest are ligonucleotidetemplated reactions that can be monitored with high sensitivity by the appearance/disappearance of a fluorescent signal upon binding to the oligonucleotide target [15-17]. Representative examples of such technologies include the use of fluorogenic probes (e.g. molecular beacons [18,19], or rely on fluorogenic reactions of chemical ligation  or primer extension . Most recent reports are based on the Staudinger reaction [22-27] diamine-catalysed aldol condensation  organomercury-activated  or SNAr reactions . Among these examples, a small proportion only was sucessfully applied for the detection of SNPs in vitro. Briefly, two modified oligonucleotides (or oligonucleotide analogues) are designed so that they can hybridize in a sequence-specific manner to a unique nucleic acid template through Watson-Crick base pairing.Upon hybridization of the synthetic probes to the complementary DNA strand, both fluorogenic moieties are brought in close enough proximity to react with each other, thus generating the fluorescent product. In case of a partial or uncomplete hybridization of one of the modified oligonucleotides (e.g. as a consequence of a SNP), both probes are no longer positioned favorably to react with each other, thus leading to a significant decrease of reaction efficiency (i.e. a weaker fluorescence). Within such systems, genetic information can be directly linked to the appearance of a characteristic fluorescence signal.
Materials and Methods
Sensing experimentsStock solutions (50 μM) of Pna1, Pna2 and DNA were prepared in water. In a typical experiment, 20 μL of a potassium phosphate buffer solution (100 mM, pH = 7.4) and 10 μL of each stock solution were transferred into one well to make the final concentrations of 10 μM Pna1, 10 μM Pna2, 10 μM DNA and 40 mM buffer. Reaction time-courses were determined in a 384-well plate using a SpectraMax M5 fluorescence plate reader (Molecular devices, UK). Reactions were carried out at 37°C with λexc = 540 nm and λem = 573 nm.
Results and DiscussionThe ability of Pna1 and Pna2 to react with each other in the absence and in the presence of various DNA sequences used as templates was investigated by fluorescence spectroscopy by monitoring the formation of a symmetrical trimethine cyanine dye (λexc = 540 nm, λem = 573 nm). Reactions were carried out in potassium phosphate buffer (40 mM, pH 7.4) at 37°C using a stoichiometric mixture of Pna1, Pna2 and DNA (10 μM each, total volume 50 μL) and the efficiency of the fluorogenic reaction was monitored over three hours in a fluorescence microplate reader. Importantly, no fluorescence was ever detected when incubating Pna1 and Pna2 under such conditions of concentration and pH and in the absence of any DNA template (Figure 3, grey curve).
|aUnderlined sequences are that complementary to Pna1 and Pna2;bXrepresents A, T, C or G.|
ConclusionIn conclusion, we have reported the use of a fast, versatile, biocompatible and fluorogenic DNA-templated reaction of cyanine dye formation for the detection of SNPs in vitro. The system offers the advantage of a reasonably high sensitivity due to the high absorptivity and moderately high fluorescence quantum yield of the cyanine dye formed  and requires minimal amounts (500 pmol) of both DNA and PNA probes when the reaction is carried out in a 384-well microtiter plate. The design of optimized (e.g. more rigid, multicoloured) systems to further improve the sensitivity of the detection is currently underway in our laboratory.
- Collins FS, Green ED, Guttmacher AE, Guyer MS (2003) A vision for the future of genomics research. Nature 422: 835-847.
- Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, et al. (2001) The sequence of the human genome. Science 291: 1304-1351.
- Stankiewicz P, Lupski JR (2010) Structural variation in the human genome and its role in disease. Annu Rev Med 61: 437-455.
- Feuk L, CarsonAR, Scherer SW (2006) Structural variation in the human genome. Nat Rev Genet 7: 85-97.
- Altshuler D, Pollara VJ, Cowles CR, Van Etten WJ, Baldwin J, et al. (2000) An SNP map of the human genome generated by reduced representation shotgun sequencing. Nature 407: 513-516.
- Halushka MK, Fan JB, Bentley K, Hsie L, Shen N, et al. (1999) Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet 22: 239-247.
- Imyanitov EN (2009) Gene polymorphisms, apoptotic capacity and cancer risk. Human Genet 125: 239-246.
- Frazer KA, Ballinger DG, Cox DR, Hinds DA, Stuve LL, et al.(2007) A second generation human haplotype map of over 3.1 million SNPs. Nature 449: 851-861.
- Kruglyak L, Nickerson DA (2001)Variation in the spice of life. Nat Genet 27: 234-236.
- Ding C, Jin S (2009) High-throughput methods for SNP genotyping. Methods Mol Biol 578: 245-254.
- LaFramboise T (2009) Single nucleotide polymorphism arrays: a decade of biological, computational and technological advances. Nucleic Acids Res 37: 4181-4193.
- Kim S, Misra A (2007) SNP genotyping: technologies and biomedical applications. Annu Rev Biomed Eng 9: 289-320.
- Percivalle C, Bartolo JF, Ladame S (2013) Oligonucleotide-templated chemical reactions: pushing the boundaries of a nature-inspired process. Org Biomol Chem 11: 16-26.
- Gorska K, Winssinger N (2013) Reactions templated by nucleic acids: more ways to translate oligonucleotide-based instructions into emerging function. Angew Chem Int Ed Engl 52: 6820-6843.
- Shibata A, Uzawa T, Nakashima Y, Ito M, Nakano Y, et al. (2013) Very rapid DNA-templated reaction for efficient signal amplification and its steady-state kinetic analysis of the turnover cycle. J Am Chem Soc 135: 14172-14178.
- Sadhu KK, Winssinger N (2013) Detection of miRNA in live cells by using templated RuII-catalyzed unmasking of a fluorophore. Chemistry 19: 8182-8189.
- Ranasinghe RT, Brown T (2005) Fluorescence based strategies for genetic analysis. Chem Commun 5487-5502.
- Venkatesan N, SeoYJ, Kim BH (2008) Quencher-free molecular beacons: a new strategy in fluorescence based nucleic acid analysis. Chem Soc Rev 37: 648-663.
- Marras SA, Kramer, FR, Tyagi S (2003) Genotyping SNPs with molecular beacons. Methods Mol Biol 212: 111-128.
- Toubanaki DK, Christopoulos TK, Ioannou PC, Flordellis CS (2009) Identification of single-nucleotide polymorphisms by the oligonucleotide ligation reaction: a DNA biosensor for simultaneous visual detection of both alleles. Anal Chem 81: 218-224.
- Krieg A, Laib S, Ruckstuhl T, Seeger S (2004) Fast detection of single nucleotide polymorphisms (SNPs) by primer elongation with monitoring of supercritical-angle fluorescence. Chem Bio Chem 5: 1680-1685.
- Cai J, Li X, Yue X, Taylor JS (2004) Nucleic acid-triggered fluorescent probe activation by the Staudinger reaction. J Am Chem Soc 126: 16324-16325.
- Pianowski ZL, Winssinger N (2007) Fluorescence-based detection of single nucleotide permutation in DNA via catalytically templated reaction. Chem Commun 3820-3822.
- Franzini RM, Kool ET (2008) 7-Azidomethoxy-coumarins as profluorophores for templated nucleic acid detection. Chembiochem 9: 2981-2988.
- Pianowski Z, Gorska K, Oswald L, Merten CA, Winssinger N (2009) Imaging of mRNA in live cells using nucleic acid-templated reduction of azidorhodamine probes. J Am Chem Soc131: 6492-6497.
- Furukawa K, Abe H, Hibino K, Sako Y, Tsuneda S, et al. (2009) Reductiontriggered fluorescent amplification probe for the detection of endogenous RNAs in living human cells. Bioconjug Chem 20:1026-1036.
- Saneyoshi S, Ito Y, Abe H (2013) Long-lived luminogenic probe for detection of RNA in a crude solution of living bacterial cells. J Am Chem Soc 135: 13632-13635.
- Huang Y, Coull JM (2008) Diamine catalyzed hemicyanine dye formation from nonfluorescent precursors through DNA programmed chemistry. J Am Chem Soc130: 3238-3239.
- Franzini RM, Kool ET (2008) Organometallic activation of a fluorogen for templated nucleic acid detection.Org Lett 10: 2935-2938.
- Shibata A, Abe H, Ito M, Kondo Y, Shimizu S, et al. (2009) DNA templated nucleophilic aromatic substitution reactions for fluorogenic sensing of oligonucleotides. Chem Commun 6586-6588.
- Meguellati K, Koripelly G, Ladame S (2010) DNA-templated synthesis of trimethine cyanine dyes: a versatile fluorogenic reaction for sensing G-quadruplex formation. Angew Chem Int Ed 49: 2738-2742.
- Koripelly G, Meguellati K, Ladame S (2010) Dual sensing of hairpin and quadruplex DNA structures using multicolored peptide nucleic acid fluorescent probes. Bioconjugchem 21: 2103-2109.
- Benson RC, Kues HA (1977) Absorption and fluorescence properties of cyanine dyes. J Chem Eng Data 22: 379-383.