Abstract
A novel approach is presented that increases sensitivity and specificity for detecting minimal traces of DNA in liquid and on solid samples. Förster Resonance Energy Transfer (FRET) from YOYO to Ethidium Bromide (EtBr) substantially increases the signal from DNA-bound EtBr highly enhancing sensitivity and specificity for DNA detection. The long fluorescence lifetime of the EtBr acceptor, when bound to DNA, allows for multi-pulse pumping with time gated (MPPTG) detection, which highly increases the detectable signal of DNA-bound EtBr. A straightforward spectra/image subtraction eliminates sample background and allows for a huge increase in the overall detection sensitivity. Using a combination of FRET and MPPTG detection an amount as small as 10 pg of DNA in a microliter sample can be detected without any additional sample purification/manipulation or use of amplification technologies. This amount of DNA is comparable to the DNA content of a one to two human cells. Such a detection method based on simple optics opens the potential for robust, highly sensitive DNA detection/imaging in the field, quick evaluation/sorting (i.e., triaging) of collected DNA samples, and can support various diagnostic assays.
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Abbreviations
- EtBr:
-
Ethidium bromide
- PBS:
-
Phosphate buffered saline
References
Betzig E, Chichester RJ (1993) Single molecules observed by near-field scanning optical microscopy. Science 262:1422–1425. https://doi.org/10.1126/science.262.5138.1422
Ceresa L, Kitchner E, Chavez J, Seung M, Bus M, Budowle B, Gryczynski I, Gryczynski Z (2021) A novel approach to imaging and visualization of minute amounts of DNA in small volume samples. Analyst 146(21):6520–6527. https://doi.org/10.1039/D1AN01391B
Ceresa L, Chavez J, Bus M, Budowle B, Kitchner E, Kimball J, Gryczynski I, Gryczynski Z (2022a) Förster resonance energy transfer-enhanced detection of minute amounts of DNA. Anal Chem. https://doi.org/10.1021/acs.analchem.1c05275
Ceresa L, Chavez J, Kitchner E, Kimball J, Gryczynski I, Gryczynski Z (2022b) Imaging and detection of long-lived fluorescence probes in presence of highly emissive and scattering background. Exp Biol Med. https://doi.org/10.1177/15353702221112121
Cihlar JC, Kapema KB, Budowle B (2022) Validation of the applied biosystems RapidHIT ID Instrument and ACE globalfiler express sample cartridge. Int J Leg Med 136(1):13–41. https://doi.org/10.1007/s00414-021-02722-9
Dziak R, Peneder A, Buetter A, Hageman C (2018) Trace DNA sampling success from evidence items commonly encountered in forensic casework. J Forensic Sci 63:835–841. https://doi.org/10.1111/1556-4029.13622
Efcavitch JW, Thompson JF (2010) Single-molecule DNA analysis. Annu Rev Anal Chem 3:109–128. https://doi.org/10.1146/annurev.anchem.111808.073558
Farka Z, Mickert MJ, Pastucha M, Mikusova Z, Skladal P, Gorris HH (2020) Advances in optical single-molecule detection: en route to supersensitive bioaffinity assays. Angew Chem Int Ed 59:10746–10773. https://doi.org/10.1002/anie.201913924
Frangioni JV (2009) The problem is background, not signal. Mol Imaging 8(6):303–304
Fudala R, Rich RM, Kimball J, Gryczynski I, Raut S, Borejdo J, Stankowska DL, Krishnamoorthy RR, Gryczynski K, Maliwal BP, Gryczynski Z (2014) Multiple-pulse pumping with time-gated detection for enhanced fluorescence imaging in cells and tissue. Springer Ser Fluoresc 1–15:225–239. https://doi.org/10.1007/4243_2014_68
Hennessy LK, Mehendale N, Chear K, Jovanovich S, Williams S, Park C, Gangano S (2014) Developmental validation of the GlobalFiler express kit, a 24-marker STR assay, on the RapidHIT system. Forensic Sci Int Genet 13:247–258. https://doi.org/10.1016/j.fsigen.2014.08.011
Kanokwongnuwut P, Martin B, Taylor D, Kirkbride KP, Linacre A (2021) How many cells are required for successful DNA profiling? Forensic Sci Int Genet 51:102453. https://doi.org/10.1016/j.fsigen.2020.102453
Keller RA, Ambrose WP, Arias AA, Cai H, Emory SR, Goodwin PM, Jett JH (2002) Analytical applications of single-molecule detection. Anal Chem 74(11):316A-324A. https://doi.org/10.1021/ac022035i
Kimball JD, Maliwal B, Raut S, Doan H, Nurekeyev Z, Gryczynski I, Gryczynski Z (2018) Enhanced DNA detection using a multiple pulse pumping scheme with time-gating (MPPTG). Analyst 143(12):2819–2827. https://doi.org/10.1039/C8AN00136G
Kitchner E, Chavez J, Ceresa L, Bus M, Budowle B, Gryczynski Z (2021) A novel approach for visualization and localization of small amounts of DNA on swabs to improve DNA collection and recovery process. Analyst 146(4):1198–1206. https://doi.org/10.1039/D0AN02043E
Lakowicz JR (2006) Principle of fluorescence spectroscopy, 3rd edn. Springer Science, New York
Li H, Ying L, Green JJ, Balasubramanian S, Klenerman D (2003) Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal Chem 75:1664–1670. https://doi.org/10.1021/ac026367z
Matveeva EG, Gryczynski Z, Stewart DR, Gryczynski I (2010) Ratiometric FRET-based detection of DNA and micro-RNA on the surface using TIRF detection. J Lumin 130:698–702. https://doi.org/10.1016/jjlumin200911022
Moerner WE, Fromm DP (2003) Methods of single-molecule fluorescence spectroscopy and microscopy. Rev Sci Instrum 74:3597–3619. https://doi.org/10.1063/1.1589587
Moerner WE, Kador L (1989) Optical detection and spectroscopy of single molecules in a solid. Phys Rev Lett 62:2535. https://doi.org/10.1103/PhysRevLett.62.2535
Murata SI, Kusba J, Piszczek G, Gryczynski I, Lakowicz JR (2000) Donor fluorescence decay analysis for energy transfer in double-helical DNA with various acceptor concentrations. Biopolymers (bio Spectroscopy) 57:306–315. https://doi.org/10.1002/1097-0282(2000)57:5%3c306::AID-BIP70%3e3.0.CO;2-7
Neely RK, Deen J, Hofkens J (2011) Optical mapping of DNA: Single-molecule-based methods for mapping genomes. Biopolymers 95:298–311. https://doi.org/10.1002/bip.21579
Rich RM, Stankowska DL, Maliwal BP, Sorensen TJ, Laursen BW, Krishnamoorthy RR, Gryczynski Z, Borejdo J, Gryczynski I, Fudala R (2013) Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore. Anal Bioanal Chem 405:2065–2075. https://doi.org/10.1007/s00216-012-6623-1
Rich RM, Gryczynski I, Fudala R, Borejdo J, Stankowska DL, Krishnamoorthy RR, Raut S, Maliwal BP, Shumilov D, Doan H, Gryczynski Z (2014) Multiple-pulse pumping for enhanced fluorescence detection and molecular imaging in tissue. Methods 66:292–298. https://doi.org/10.1016/j.ymeth.2013.08.026
Rich RM, Gryczynski I, Borejdo J, Gryczynski Z (2017) Multi-Pulse Pumping for Enhanced Fluorescence Detection and Molecular Imaging in Cells and Tissue. US Patent # US 9612245 B2
Shumilov D, Rich RM, Gryczynski I, Raut S, Gryczynski K, Kimball J, Doan H, Sørensen TJ, Laursen BW, Borejdo J, Gryczynski Z (2014) Generating multiple-pulse bursts for enhanced fluorescence detection. Methods Appl Fluoresc. https://doi.org/10.1088/2050-6120/2/2/024009
Singh-Zocchi M, Dixit S, Zocchi G (2003) Single-molecule detection of DNA hybridization. PNAS 100:7605–7610. https://doi.org/10.1073/pnas.1337215100
Van Oorschot RAH, Ballantyne KN, Mitchell RJ (2010) Forensic trace DNA: a review. Investigative Genet 1:14. https://doi.org/10.1186/2041-2223-1-14
Zhao X, Tapec-Dytioco R, Weihong T (2003) Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles. JACS 125:11474–11475
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The authors have declared that no conflicting interests exist. Z. Gryczynski acknowledges support from “Tex” Moncrief Jr. and TCU College of Science and Engineering.
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The manuscript was written through contributions of all authors. LC and ZG wrote the manuscript. LC and JC performed the experiments. MB, BB, IG, EK and JK contributed to the manuscript review.
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Ceresa, L., Chavez, J., Bus, M.M. et al. Multi intercalators FRET enhanced detection of minute amounts of DNA. Eur Biophys J 52, 593–605 (2023). https://doi.org/10.1007/s00249-023-01655-y
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DOI: https://doi.org/10.1007/s00249-023-01655-y