Abstract
Due to its incredible sensitivity, polymerase chain reaction (PCR) is a method of choice for nucleic acid diagnostics. However, PCR is a complex reaction due to (1) limited yield of product DNA (PCR plateau), (2) temperature cycling, and (3) complicated quantification methods. We have developed quadruplex priming amplification (QPA) to greatly simplify nucleic acid amplification and real-time quantification assays. The method relies on specifically designed guanine-rich primers, which after polymerase elongation are capable of spontaneous dissociation from target sites and forming DNA quadruplex. The quadruplex is characterized by significantly more favorable thermodynamics than the corresponding DNA duplexes. As a result, target sequences are accessible for the next round of priming, and DNA amplification proceeds under isothermal conditions with improved product yield. In addition, the quadruplex formation is accompanied by an increase in intrinsic fluorescence of the primers, allowing simple and accurate detection of product DNA. The chapter discusses the thermodynamic and optical principles of QPA and its application in nucleic acid diagnostics.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Adams NM, Wang KK, Caprioli AC et al (2014) Quadruplex priming amplification for the detection of mRNA from surrogate patient samples. Analyst 139:1644–1652
Bordelon H, Adams NM, Klemm AS et al (2011) Development of a low-resource RNA extraction cassette based on surface tension valves. ACS Appl Mater Interfaces 3:2161–2168
Cheng X, Liu X, Bing T et al (2009) General peroxidase activity of G-quadruplex-hemin complexes and its application in ligand screening. Biochemistry 48:7817–7823
Edwards KJ, Logan JMJ (2009) Peforming real-time PCR. In: Logan J et al (eds) Real-time PCR. Caister Academic, Norfolk, pp 85–93
Gogichaishvili S, Johnson J, Gvarjaladze D et al (2014a) Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin. Biopolymers 101:583–590
Gogichaishvili S, Lomidze L, Kankia B (2014b) Quadruplex priming amplification combined with nicking enzyme for diagnostics. Anal Biochem 466:44–48
Hawkins ME (2007) Synthesis, purification and sample experiment for fluorescent pteridine-containing DNA: tools for studying DNA interactive systems. Nat Protoc 2:1013–1021
Hawkins ME (2008) Fluorescent pteridine probes for nucleic acid analysis. Methods Enzymol 450:201–231
Hawkins ME, Pfleiderer W, Balis FM et al (1997) Fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into oligonucleotides. Anal Biochem 244:86–95
Holland PM, Abramson RD, Watson R et al (1991) Detection of specific polymerase chain reaction product by utilizing the 5′––3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280
Johnson J, Okyere R, Joseph A et al (2013) Quadruplex formation as a molecular switch to turn on intrinsically fluorescent nucleotide analogs. Nucleic Acids Res 41:220–228
Kankia BI (2004) Optical absorption assay for strand-exchange reactions in unlabeled nucleic acids. Nucleic Acids Res 32:e154
Kankia BI (2006) A real-time assay for monitoring nucleic acid cleavage by quadruplex formation. Nucleic Acids Res 34:e141
Kankia BI (2011) Self-dissociative primers for nucleic acid amplification and detection based on DNA quadruplexes with intrinsic fluorescence. Anal Biochem 409:59–65
Kourentzi KD, Fox GE, Willson RC (2003) Hybridization-responsive fluorescent DNA probes containing the adenine analog 2-aminopurine. Anal Biochem 322:124–126
Law SM, Eritja R, Goodman MF et al (1996) Spectroscopic and calorimetric characterizations of DNA duplexes containing 2-aminopurine. Biochemistry 35:12329–12337
Lee MA, Squirrell DJ, Leslie DL et al (2009) Homogeneous fluorescent chemistries for real-time PCR. In: Logan J et al (eds) Real-time PCR. Caister Academic, Norfolk, pp 23–45
Loh Q, Omar N, Glokler J et al (2014) IQPA: isothermal nucleic acid amplification-based immunoassay using DNAzyme as the reporter system. Anal Biochem 463:67–69
Marti AA, Jockusch S, Li Z et al (2006) Molecular beacons with intrinsically fluorescent nucleotides. Nucleic Acids Res 34:e50
McLaughlin LW, Leong T, Benseler F et al (1988) A new approach to the synthesis of a protected 2-aminopurine derivative and its incorporation into oligodeoxynucleotides containing the Eco RI and Bam HI recognition sites. Nucleic Acids Res 16:5631–5644
Niemz A, Ferguson TM, Boyle DS (2011) Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 29:240–520
Partskhaladze T, Taylor A, Lomidze L et al (2015) Exponential quadruplex priming amplification for DNA-based isothermal diagnostics. Biopolymers 103:88–95
Pfaffl MW, Vandesompele J, Kubitsa M (2009) Data analysis software. In: Logan J et al (eds) Real-time PCR. Caister Academic, Norfolk, pp 65–83
Taylor A, Joseph A, Okyere R et al (2013) Isothermal quadruplex priming amplification for DNA-based diagnostics. Biophys Chem 171:1–8
Travascio P, Bennet AJ, Wang DY et al (1999) A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites. Chem Biol 6:779–787
Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14:303–308
Tyagi S, Bratu DP, Kramer FR (1998) Multicolor molecular beacons for allele discrimination. Nat Biotechnol 16:49–53
Van Ness J, Van Ness LK, Galas DJ (2003) Isothermal reactions for the amplification of oligonucleotides. Proc Natl Acad Sci U S A 100:4504–4509
Whitcombe D, Theaker J, Guy SP et al (1999) Detection of PCR products using self-probing amplicons and fluorescence. Nat Biotechnol 17:804–807
Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415
Acknowledgments
This work was funded by a grant (DI/23/7-230/12) from Shota Rustaveli National Science Foundation (Republic of Georgia) and a grant from the Bill & Melinda Gates Foundation through the Grand Challenges in Global Health initiative.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer International Publishing Switzerland
About this chapter
Cite this chapter
Kankia, B. (2015). Quadruplex Priming Amplification (QPA) for Nucleic Acid Diagnostics. In: Erdmann, V., Jurga, S., Barciszewski, J. (eds) RNA and DNA Diagnostics. RNA Technologies. Springer, Cham. https://doi.org/10.1007/978-3-319-17305-4_14
Download citation
DOI: https://doi.org/10.1007/978-3-319-17305-4_14
Publisher Name: Springer, Cham
Print ISBN: 978-3-319-17304-7
Online ISBN: 978-3-319-17305-4
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)