Hostname: page-component-5c6d5d7d68-pkt8n Total loading time: 0 Render date: 2024-08-15T08:31:21.637Z Has data issue: false hasContentIssue false
  • English
  • Français

Resilience of DNA chains to molecular fracture after PCR heating cycles and implications on PCR reliability

Published online by Cambridge University Press:  15 August 2024

Roberto Serpieri Open the ORCID record for Roberto Serpieri [Opens in a new window]  and
Fabio Franchi Open the ORCID record for Fabio Franchi [Opens in a new window]
Roberto Serpieri*
Affiliation:
Dipartimento di Architettura e Disegno Industriale, Universita della Campania “Luigi Vanvitelli,” Aversa, Italy
Fabio Franchi
Affiliation:
Independent Researcher, Trieste, Italy
*
Corresponding author: Roberto Serpieri; Email: roberto.serpieri@unicampania.it
Get access Rights & Permissions [Opens in a new window]

Abstract

Soon after its introduction in 1987, polymerase chain reaction (PCR) has become a technique widely employed in diagnostic medical devices and forensic science with the intention of amplifying genetic information. PCR prescribes that each of its cycles must include a heating subprocess at 95 °C or more (denominated DNA denaturation and provided for allowing a claimed orderly separation of the two complementary nucleotides strands), which can produce significant damage to DNA, caused by high-speed collisions with surrounding molecules. Since such disruption should be prevented in order to reliably employ PCR, a study of the mechanics of such loss of structural integrity is herein presented, preceded by a review of the fundamental literature which has elucidated the effects of molecular agitation on DNA fragmentation. The main conclusion of this retrospective survey is that the body of examined theoretical and experimental evidence consistently and redundantly confirms scarce resilience and significant loss of structural integrity when DNA is heated at temperatures above 90 °C, even for 1 minute. Such conclusion contradicts the claimed paradigm of PCR fidelity and raises the concern that, at least for long sequences, if PCR can amplify some information, such amplified information may be unreliable for diagnostic or forensic applications, since it originates from sequences of nucleotides subjected to random fragmentation and reaggregation. Such a low-reliability scenario should be preventively considered in the various fields where DNA amplification methodologies are employed which provide for high-temperature heating under conditions equal to or similar to those prescribed by the PCR protocols reviewed in this study.

Keywords

DNADNA annealingDNA denaturationNAATnano-molecular structuresphysical chemistry
Type
Review
Information
Quarterly Reviews of Biophysics , Volume 57 , 2024 , e8
Copyright
© The Author(s), 2024. Published by Cambridge University Press

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Air, GM, Sanger, F and Coulson, AR (1976) Nucleotide and amino acid sequences of gene G of ϕX174. Journal of Molecular Biology 108, 519–533.CrossRefGoogle Scholar
Alexander, P and Stacey, KA (1955) Evidence from light-scattering studies for a dimeric structure for deoxyribonucleic acid in solution. Biochemical Journal 60(2), 194.CrossRefGoogle ScholarPubMed
Alexander, P and Stacey, KA (1955) The light-scattering and non-Newtonian viscosity of high molecular weight polymethacrylic acid. Transactions of the Faraday Society 51, 299–308.CrossRefGoogle Scholar
Anonymous (1989) DNA amplification. Nature 341, 570. https://doi.org/10.1038/341570a0.CrossRefGoogle Scholar
Applequist, J (1961) A model for random degradation of two-stranded polymers and its application to deoxyribonucleic acid. Archives of Biochemistry and Biophysics 95(1), 42–54.CrossRefGoogle Scholar
Barrow, GM (1966) Physical Chemistry, 2nd Edn. New York, NY; Tokyo, Japan: McGraw-Hill; Kōgakusha.Google Scholar
Blout, ER and Asadourian, A (1954) The ultraviolet absorption spectra of desoxypentose nucleic acid. Biochimica et Biophysica Acta 13, 161–170.CrossRefGoogle ScholarPubMed
Butler, JAV, Shooter, KV and Pain, RH (1956) The sedimentation behaviour of DNA and the effect of heat and X-rays. In Proceedings of the Third International Congress of Biochemistry, Brussels, 1–6 August. New York: Academic Press, p. 141.Google Scholar
Carr, CI and Zimm, BH (1950) Absolute intensity of light scattering from pure liquids and solutions. The Journal of Chemical Physics 18(12), 1616–1626.CrossRefGoogle Scholar
Caton, AJ and Robertson, JS (1979) New procedure for the production of influenza virus-specific double-stranded DNA’s. Nucleic Acids Research 7(6), 1445–1456.CrossRefGoogle ScholarPubMed
Cavalieri, LF and Angelos, A (1950) Studies on the structure of nucleic acids. I. Interaction of rosaniline with desoxypentose nucleic acid. Journal of the American Chemical Society 72(10), 4686–4690.CrossRefGoogle Scholar
Cavalieri, LF and Rosenberg, BH (1957) Studies on the structure of nucleic acids. XI. The roles of heat and acid in deoxyribonucleic acid denaturation. Journal of the American Chemical Society 79(20), 5352–5357.CrossRefGoogle Scholar
Cecil, R and Ogston, AG (1948a) The accuracy of the Svedberg oil-turbine ultracentrifuge. Biochemical Journal 43(4), 592.CrossRefGoogle ScholarPubMed
Cecil, R and Ogston, AG (1948b) The sedimentation of thymus nucleic acid in the ultracentrifuge. Journal of the Chemical Society 280, 1382–1386.CrossRefGoogle Scholar
Chargaff, E (1955) Isolation and composition of the Deoxypentose nucleic acids and of the corresponding nucleoproteins. In Chargaff, A, Davidson, JN (eds.), The Nucleic Acids, Vol. 1, 2nd prn. London: Academic Press, pp. 307–368.Google Scholar
Chargaff, E (1968) What really is DNA? Remarks on the changing aspects of a scientific concept. Progress in Nucleic Acid Research and Molecular Biology 8, 297–333.CrossRefGoogle ScholarPubMed
Charlesby, A (1954) Molecular-weight changes in the degradation of long-chain polymers. Proceedings of the Royal Society of London. Series A Mathematical and Physical Sciences 224(1156), 120–128.Google Scholar
Conway, BE and Butler, JAV (1952) The action of denaturing agents on deoxyribonucleic acid. Journal of the Chemical Society 590, 3075–3082.CrossRefGoogle Scholar
Cox, RA, Overend, WG, Peacocke, AR and Wilson, S (1955) Effects of gamma-rays on solutions of sodium deoxyribonucleate: possible chemical mechanism for their biological effects. Nature 176(4489), 919–921. https://doi.org/10.1038/176919a0.CrossRefGoogle Scholar
Cox, RA, Overend, WG, Peacocke, AR and Wilson, S (1958) The action of γ-rays on sodium deoxyribo-nucleate in solution. Proceedings of the Royal Society of London. Series B-Biological Sciences 149(937), 511–533.Google ScholarPubMed
Cox, RA and Peacocke, AR (1956) Electrometric titration of the sodium salts of deoxyribonucleic acids. Part IV. Denaturation by heat in aqueous solution. Journal of the Chemical Society, 2646–2651.CrossRefGoogle Scholar
Cox, RA and Peacocke, AR (1957) Application of the titration method to studies on the denaturation of sodium deoxyribonucleate. Journal of Polymer Science 23(104), 765–779.CrossRefGoogle Scholar
Creeth, JM, Gulland, JM and Jordan, DO (1947) Deoxypentose nucleic acids. Part III. Viscosity and streaming birefringence of solutions of the sodium salt of the deoxypentose nucleic acid of calf thymus. Journal of the Chemical Society 214, 1141–1145.CrossRefGoogle Scholar
Debye, P (1944) Light scattering in solutions. Journal of Applied Physics 15(4), 338–342.CrossRefGoogle Scholar
Debye, PP (1946) A photoelectric instrument for light scattering measurements and a differential refractometer. Journal of Applied Physics 17(5), 392–398.CrossRefGoogle Scholar
Debye, P (1947) Molecular-weight determination by light scattering. The Journal of Physical Chemistry 51(1), 18–32.CrossRefGoogle ScholarPubMed
Dekker, CA and Schachman, HK (1954) On the macromolecular structure of deoxyribonucleic acid: An interrupted two-strand model. Proceedings of the National Academy of Sciences of the United States of America 40(10), 894.CrossRefGoogle ScholarPubMed
Doty, P and Bunce, BH (1952) The molecular weight and shape of desoxypentose nucleic acid. Journal of the American Chemical Society 74(20), 5029–5034.CrossRefGoogle Scholar
Doty, P, Marmur, J, Eigner, J and Schildkraut, C (1960) Strand separation and specific recombination in deoxyribonucleic acids: Physical chemical studies. Proceedings of the National Academy of Sciences of the United States of America 46(4), 461–476.CrossRefGoogle ScholarPubMed
Doty, P and Rice, SA (1955) The denaturation of desoxypentose nucleic acid. Biochimica et Biophysica Acta 16, 446–448.CrossRefGoogle ScholarPubMed
European Bioinformatics Institute (2021) Universal Protein Resource, Uni-ProtKB – P59594 (SPIKE_SARS). Available at https://www.uniprot.org/uniprot/P59594#PRO_0000037208 (accessed 29 October 2021).Google Scholar
Felsenfeld, G, Davies, DR and Rich, A (1957) Formation of a three-stranded polynucleotide molecule. Journal of the American Chemical Society 79, 2023–2024.CrossRefGoogle Scholar
Freund, A, Pouyet, J and Sadron, C (1958) Action de la chaleur Sur lacide desoxyribonucleique-variations de la masse des particules en solution. Comptes Rendus Hebdomadaires des Séances de l’Académie des Sciences 246(8), 1306–1309.Google Scholar
Gaubatz, J and Paddock, GV (1982) Strategies for constructing complementary DNA for cloning. Journal of Theoretical Biology 95(4), 679–696.CrossRefGoogle ScholarPubMed
Geiduschek, P and Holtzer, A (1959) Application of light scattering to biological systems: Deoxyribonucleic acid and the muscle proteins. Advances in Biological and Medical Physics 6, 431–551.CrossRefGoogle Scholar
Goldstein, G and Stern, KG (1950) Experiments on the sonic, thermal, and enzymic depolymerization of desoxyribosenucleic acid. Journal of Polymer Science 5(6), 687–708.CrossRefGoogle Scholar
Hall, BD and Spiegelman, S (1961) Sequence complementarity of T2-DNA and T2-specific RNA. Proceedings of the National Academy of Sciences 47(2), 137–146.CrossRefGoogle ScholarPubMed
Hamaguchi, K and Geiduschek, EP (1962) The effect of electrolytes on the stability of the deoxyribonucleate helix. Journal of the American Chemical Society 84(8), 1329–1338.CrossRefGoogle Scholar
Innis, MA, Myambo, KB, Gelfand, DH and Brow, MA (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proceedings of the National Academy of Sciences 85(24), 9436–9440. http://doi.org/10.1073/pnas.85.24.9436.CrossRefGoogle ScholarPubMed
Jordan, DO (1955) The physical properties of nucleic acids. In Chargaff, A and Davidson, JN (eds.), The Nucleic Acids, Vol. 1, 2nd prn. London: Academic Press, pp. 447–461.Google Scholar
Josse, J, Kaiser, AD and Kornberg, A (1961) Enzymatic synthesis of deoxyribonucleic acid VIII. Frequencies of nearest neighbor base sequences in deoxyribonucleic acid. Journal of Biological Chemistry 236(3), 864–875.CrossRefGoogle ScholarPubMed
Kleppe, K, Ohtsuka, E, Kleppe, R, Molineux, I and Khorana, HG (1971) Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA’s as catalyzed by DNA polymerases. Journal of Molecular Biology 56(2), 341–361. http://doi.org/10.1016/0022-2836(71)90469-4.CrossRefGoogle ScholarPubMed
Krejci, LE, Sweeny, L and Hambleton, J (1949) Molecular weights of desoxyribonucleic acid polymers. Journal of the Franklin Institute 248(2), 177–180.CrossRefGoogle Scholar
Kurnick, NB (1954a) Mechanism of desoxyribonuclease depolymerization: Effect of physical and enzymatic depolymerization on the affinity of methyl green and of desoxyribonuclease for desoxyribonucleic acid1a. Journal of the American Chemical Society 76(2), 417–424.CrossRefGoogle Scholar
Kurnick, NB (1954b) Methyl green-pyronin I. Basis of selective staining of nucleic acids. Journal of General Physiology 33, 243.CrossRefGoogle Scholar
Kwok, S, Mack, DH, Mullis, KB, Poiesz, B, Ehrlich, G, Blair, D, Friedman-Kien, A and Sninsky, JJ (1987) Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. Journal of Virology 61(5), 1690–1694.CrossRefGoogle ScholarPubMed
Lee, WA and Peacocke, AR (1951) 742. Electrometric titration of the sodium salts of deoxyribonucleic acids. Part I. Journal of the Chemical Society (Resumed), 3361–3373.CrossRefGoogle Scholar
Loring, AS (1955) Hydrolysis of nucleic acids and procedures for the direct estimation of purine and pyrimidine fractions by absorption spectrophotometry. In Chargaff, A and Davidson, JN (eds.), The Nucleic Acids, Vol. 1, 2nd prn. London: Academic Press, pp. 191–199.Google Scholar
Mandelkern, L, Krigbaum, WR, Sheraga, HA and Flory, PJ (1952) Sedimentation behavior of flexible chain molecules: Polyisobutilene. The Journal of Chemical Physics 20, 1392–1397.CrossRefGoogle Scholar
Miyaji, T and Price, VE (1950) Protective effect of salt on reduction of viscosity of sodium thymonucleate solutions by heat. Proceedings of the Society for Experimental Biology and Medicine 75(2), 311–313.CrossRefGoogle ScholarPubMed
Mullis, KB (1985) US Patent Application No: 06/716,975, filed Mar. 28, 1985. Abandoned.Google Scholar
Mullis, KB (1987) US Patent Application No 06/791308, filed July 28, 1987. No. 4683202. Process for Amplifying Nucleic Acid Sequences.Google Scholar
Mullis, KB (1990) The unusual origin of the polymerase chain reaction. Scientific American 262(4), 56–65.CrossRefGoogle ScholarPubMed
Mullis, KB and Faloona, FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods in Enzymology 155, 335–350.CrossRefGoogle Scholar
Panet, A and Khorana, HG (1974). Studies on polynucleotides. The linkage of deoxyribopolynucleotide templates to cellulose and its use in their replication. Journal of Biological Chemistry 249(16), 5213–5221.CrossRefGoogle ScholarPubMed
Peacocke, AR and Preston, BN (1958) The degradation and denaturation of sodium deoxyribonucleate by γ-rays. Journal of Polymer Science 31(122), 1–14.CrossRefGoogle Scholar
Perry, JH, Chilton, CH, Kirkpatrick, SD, et al. (1963) Chemical Engineersʼ Handbook, Perry, RH, Chilton, CH and Kirkpatrick, SD, eds. Vol. 1963. New York: McGraw-Hill.Google Scholar
Reichmann, ME, Bunce, BH and Doty, P (1953) The changes induced in sodium desoxyribonucleate by dilute acid. Journal of Polymer Science 10(1), 109–119.CrossRefGoogle Scholar
Reichmann, ME, Rice, SA, Thomas, CA and Doty, P (1954) A further examination of the molecular weight and size of desoxypentose nucleic acid. Journal of the American Chemical Society 76(11), 3047–3053.CrossRefGoogle Scholar
Rice, SA and Doty, P (1957) The thermal denaturation of desoxyribose nucleic acid. Journal of the American Chemical Society 79(15), 3937–3947.CrossRefGoogle Scholar
Rich, A (1957) The structure of synthetic Polyribonucleotides and the spontaneous formation of a new two-stranded helical molecule. In By, WDME and Glass, B (eds.), The Chemical Basis of Heredity. Baltimore: The Johns Hopkins University Press, pp. 557–562.Google Scholar
Rich, A (1958) Formation of two- and three-stranded helical molecules by polyinosinic acid and polyadenylic acid. Nature 181(4608), 521–525. http://doi.org/10.1038/181521a0.CrossRefGoogle ScholarPubMed
Rich, A (2006) Discovery of the hybrid helix and the first hybridization. The Journal of Biological Chemistry 281(12), 7693–7696.CrossRefGoogle ScholarPubMed
Rich, A and Davies, DR (1956) A new two stranded helical structure: Polyadenylic acid and polyuridylic acid. Journal of American Chemistry Society 78(14), 3548–3549. http://doi.org/10.1021/ja01595a086.CrossRefGoogle Scholar
Rowen, JW and Norman, A (1954) The size of deoxyribonucleic acid molecules in E. coli. Archives of Biochemistry and Biophysics 51(2), 524–528.CrossRefGoogle ScholarPubMed
Sadron, C (1955) Essai d’interprétation de quelques propriétés physico-chimiques des solutions étendue d’acide désoxyribonucléique. In Proceedings of the Third International Congress of Biochemistry: Brussels. New York: Academic Press, pp. 125–135.Google Scholar
Sadron, C (1959) Les propriétés physiques de l’acide désoxyribonucléique en solution. In Les nucléo-protéines. Actes de la onzième Conseil de Chimie tenu à l’Université libre de Bruxelles du 1er au 6 juin 1959, Institut International de Chimie Solvay, Brussels, 1–6 June. New York: Interscience Publishers, p. 105.Google Scholar
Saiki, RK, Scharf, S, Faloona, F, Mullis, KB, Horn, GT, Erlich, HA and Arnheim, N (1985) Enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230(4732), 1350–1354.CrossRefGoogle ScholarPubMed
Sanger, F and Coulson, AR (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. Journal of Molecular Biology 94(3), 441–448.CrossRefGoogle ScholarPubMed
Sanger, F, Nicklen, S and Coulson, AR (1977) DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences 74(12), 5463–5467.CrossRefGoogle ScholarPubMed
Scharf, SJ, Horn, GT and Erlich, HA (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233(4768), 1076–1078.CrossRefGoogle ScholarPubMed
Schumaker, VN, Richards, EG and Schachman, HK (1956) A study of the kinetics of the enzymatic digestion of deoxyribonucleic acid. Journal of the American Chemical Society 78(17), 4230–4236.CrossRefGoogle Scholar
Shooter, KV and Butler, JAV (1955) Apparent heterogeneity of deoxyribonucleic acid: Sedimentation experiments at low concentrations. Nature 175(4455), 500–502.CrossRefGoogle ScholarPubMed
Shooter, KV, Pain, RH and Butler, JAV (1956) The effect of heat and x-rays on deoxyribonucleic acid. Biochimica et Biophysica Acta 20, 497–502.CrossRefGoogle ScholarPubMed
Simha, R (1949) Effect of concentration on the viscosity of dilute solutions. Journal of Research of the National Bureau of Standards 42, 409.CrossRefGoogle ScholarPubMed
Svedberg, T (1938) The ultracentrifuge and its field of research. Industrial & Engineering Chemistry Analytical Edition 10(3), 113–127.CrossRefGoogle Scholar
Tamm, C and Chargaff, E (1953) Physical and chemical properties of the apurinic acid of calf thymus. Journal of Biological Chemistry 203(2), 689–694.CrossRefGoogle ScholarPubMed
Thomas, R (1951) Sur l’existence, dans la molécule des acides nucléiques, d’une structure secondaire à liaisons labiles. Experientia 7(7), 261–262.CrossRefGoogle Scholar
Thomas, R (1954a) Recherches Sur la dénaturation des acides desoxyribonucléiques. Biochimica et Biophysica Acta 14, 231–240.CrossRefGoogle Scholar
Thomas, R (1954b) The denaturation of desoxyribonucleic acids. Transactions of the Faraday Society 50, 304.Google Scholar
Thomas, CA and Doty, P (1956) The mild acidic degradation of desoxyribose nucleic acid. Journal of the American Chemical Society 78(9), 1854–1860.CrossRefGoogle Scholar
US National Center for Biotechnology Information (2021) GenBank: QGV12784.1. Spike protein [Sus scrofa]. Available at https://www.ncbi.nlm.nih.gov/protein/QGV12784.1 (accessed 29 October 2021).Google Scholar
Watson, JD and Crick, FH (1953) Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid. Nature 171(4356), 737–738.CrossRefGoogle ScholarPubMed
Williams, RC (1952) Electron microscopy of sodium desoxyribonucleate by use of a new freeze-drying method. Biochimica et Biophysica Acta 9, 237–239.CrossRefGoogle ScholarPubMed
Wu, R and Kaiser, AD (1968) Structure and base sequence in the cohesive ends of bacteriophage lambda DNA. Journal of Molecular Biology 35(3), 523–537.CrossRefGoogle ScholarPubMed
Zamenhof, S, Alexander, HE and Leidy, G (1953) Studies on the chemistry of the transforming activity: I. Resistance to Physical and Chemical Agents. The Journal of Experimental Medicine 98(4), 373–397.CrossRefGoogle ScholarPubMed
Zamenhof, S and Chargaff, E (1950) Studies on the diversity and the native state of desoxypentose nucleic acids. Journal of Biological Chemistry 186(1), 207–219.CrossRefGoogle ScholarPubMed
Zamenhof, S, Griboff, G and Marullo, N (1954) Studies on the resistance of desoxyribonucleic acids to physical and chemical factors. Biochimica et Biophysica Acta 13, 459–470.CrossRefGoogle ScholarPubMed
Zamenhof, S, Leidy, G, Hahn, E and Alexander, HE (1956) Inactivation and unstabilization of the transforming principle by mutagenic agents. Journal of Bacteriology 72(1), 1–11.CrossRefGoogle ScholarPubMed
Zimm, BH (1948a) The scattering of light and the radial distribution function of high polymer solutions. The Journal of Chemical Physics 16(12), 1093–1099.CrossRefGoogle Scholar
Zimm, BH (1948b) Apparatus and methods for measurement and interpretation of the angular variation of light scattering; preliminary results on polystyrene solutions. The Journal of Chemical Physics 16(12), 1099–1116.CrossRefGoogle Scholar