Simplified detection of the asymmetric polymerase chain reaction-amplified DNA and its application in the target identification

G Suhasa; Savithri Bhat

doi:10.7324/JABB.2018.60208

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Abstract

The nucleic acid amplification methods such as the polymerase chain reaction (PCR) and the loop-mediated isothermal amplification rely on the detection of the amplified products by means of agarose gel electrophoresis. The detection of the amplified target DNA in an asymmetric PCR was simplified by carrying out the probe hybridization, polymerization, and the subsequent measurement of fluorescence of the double-stranded target DNA (dsDNA) using the Qubit^®dsDNA BR Assay Kit. This method was validated by detecting the lacZ gene that is present in the pUC 18 plasmid as well as by detecting the CDH13 gene that is present in the human genomic DNA.

Keyword: Polymerase chain reaction Agarose gel electrophoresis Asymmetric Loop-mediated isothermal amplification Fluorometry.

Citation:

Suhasa G, Bhat S. Simplified detection of the asymmetric polymerase chain reaction-amplified DNA and its application in the target identification. J App Biol Biotech. 2018;6(2):50-53. DOI: 10.7324/JABB.2018.60208

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

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2. Killeen AA. A visible spectrophotometric assay for submicrogram quantities of DNA, including PCR-amplified DNA. Microchemical Journal. 1995; 52(3):333-340. https://doi.org/10.1006/mchj.1995.1105
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8. Almasi MA, Erfan Manesh M, Jafary H, Dehabadi SM. Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye. Journal of Virological Methods. 2013; 192(1-2):51-54. https://doi.org/10.1016/j.jviromet.2013.04.014
9. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology. 1986; 51(1):263-73. https://doi.org/10.1101/SQB.1986.051.01.032
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13. Nolasco G, Sequeira Z, Soares C, Mansinho A, Bailey AM., Niblett CL. Asymmetric PCR ELISA: Increased Sensitivity and Reduced Costs for the Detection of Plant Viruses. European Journal of Plant Pathology. 2002; 108(4):293–298. https://doi.org/10.1023/A:1015649429160
14. Deng H, Xu Y, Liu Y, Che Z, Guo H, Shan S, Sun Y et al. Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence. Analytical Chemistry. 2012; 84(3):1253-1258. https://doi.org/10.1021/ac201713t
15. Wang L, Liu Z, Xia X, Huang J. Visual detection of Maize chlorotic mottle virus by asymmetric polymerase chain reaction with unmodified gold nanoparticles as the colorimetric probe. Analytical Methods. 2016; 8(38):6959-6964. https://doi.org/10.1039/C6AY02116F
16. Comas D, Plaza S, Calafell F, Sajantila A, Bertranpetit J. Recent insertion of an Alu element within a polymorphic human-specific Alu insertion. Molecular Biology and Evolution. 2001; 18(1):85-88. https://doi.org/10.1093/oxfordjournals.molbev.a003722
17. Sanchez JA, Pierce KE, Rice JE, Wangh LJ. Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proceedings of the National Academy of Sciences. 2004; 101(7):1933-1938. https://doi.org/10.1073/pnas.0305476101
18. Bonasera V, Alberti S, Sacchetti A. Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide. Biotechniques. 2007; 43(2):173-174. https://doi.org/10.2144/000112500

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1. Green MR, Sambrook J. Molecular Cloning a laboratory manual, 4th ed. USA: Cold Spring Harbour, 2012.

2. Killeen AA. A visible spectrophotometric assay for submicrogram quantities of DNA, including PCR-amplified DNA. Microchemical Journal. 1995; 52(3):333-340. https://doi.org/10.1006/mchj.1995.1105

3. Peters DL, Dahmus ME. A Method of DNA Quantitation for Localization of DNA in Metrizamide Gradients. Analytical Biochemistry. 1979; 93(2):306-311. https://doi.org/10.1016/S0003-2697(79)80156-6

4. Sinicropi D, Baker DL, Prince WS, Shiffer K, Shak S. Colorimetric determination of DNase I activity with a DNA-methyl green substrate. Analytical Biochemistry. 1994; 222(2):351-358. https://doi.org/10.1006/abio.1994.1502

5. Toumazou C, Shepherd LM, Reed SC, Chen GI, Patel A, Garner DM, Wang CJ et al. Simultaneous DNA amplification and detection using a pH-sensing semiconductor system. Nature Methods. 2013; 10(7):641-646. https://doi.org/10.1038/nmeth.2520

6. Tanner NA, Zhang Y, Evans TC Jr. Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes. Biotechniques. 2015; 58(2):59-68. https://doi.org/10.2144/000114253

7. Lee K, Kim K. A colorimetric confirmation method for DNA amplification in PCR and its application to the detection of Giardia lamblia cysts. Biotechnology Letters. 2003; 25(20):1739–1742. https://doi.org/10.1023/A:1026035628935

8. Almasi MA, Erfan Manesh M, Jafary H, Dehabadi SM. Visual detection of Potato Leafroll virus by loop-mediated isothermal amplification of DNA with the GeneFinder™ dye. Journal of Virological Methods. 2013; 192(1-2):51-54. https://doi.org/10.1016/j.jviromet.2013.04.014

9. Mullis K, Faloona F, Scharf S, Saiki R, Horn G, Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symposia on Quantitative Biology. 1986; 51(1):263-73. https://doi.org/10.1101/SQB.1986.051.01.032

10. Gyllensten UB, Erlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proceedings of the National Academy of Sciences. 1988; 85(20):7652-7656. https://doi.org/10.1073/pnas.85.20.7652

11. Mazars GR, Moyret C, Jeanteur P, Theillet CG. Direct sequencing by thermal asymmetric PCR. Nucleic Acids Research. 1991; 19(17):4783. https://doi.org/10.1093/nar/19.17.4783

12. Fujii T, Ohta M, Kono M, Hoshina S, Fukuhara K, Tsuruoka M. Detection of the gene of Legionella pneumophila using the fluorescence polarization with the asymmetric PCR. Nucleic Acids Symposium Series. 1999; 42(1):59-60. https://doi.org/10.1093/nass/42.1.59

13. Nolasco G, Sequeira Z, Soares C, Mansinho A, Bailey AM., Niblett CL. Asymmetric PCR ELISA: Increased Sensitivity and Reduced Costs for the Detection of Plant Viruses. European Journal of Plant Pathology. 2002; 108(4):293–298. https://doi.org/10.1023/A:1015649429160

14. Deng H, Xu Y, Liu Y, Che Z, Guo H, Shan S, Sun Y et al. Gold nanoparticles with asymmetric polymerase chain reaction for colorimetric detection of DNA sequence. Analytical Chemistry. 2012; 84(3):1253-1258. https://doi.org/10.1021/ac201713t

15. Wang L, Liu Z, Xia X, Huang J. Visual detection of Maize chlorotic mottle virus by asymmetric polymerase chain reaction with unmodified gold nanoparticles as the colorimetric probe. Analytical Methods. 2016; 8(38):6959-6964. https://doi.org/10.1039/C6AY02116F

16. Comas D, Plaza S, Calafell F, Sajantila A, Bertranpetit J. Recent insertion of an Alu element within a polymorphic human-specific Alu insertion. Molecular Biology and Evolution. 2001; 18(1):85-88. https://doi.org/10.1093/oxfordjournals.molbev.a003722

17. Sanchez JA, Pierce KE, Rice JE, Wangh LJ. Linear-after-the-exponential (LATE)-PCR: an advanced method of asymmetric PCR and its uses in quantitative real-time analysis. Proceedings of the National Academy of Sciences. 2004; 101(7):1933-1938. https://doi.org/10.1073/pnas.0305476101

18. Bonasera V, Alberti S, Sacchetti A. Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using ethidium bromide. Biotechniques. 2007; 43(2):173-174. https://doi.org/10.2144/000112500