Enhancing DNA/RNA Extraction Efficiency in Viscous Samples Using Mucolytic Agents
Keywords:
nucleic acid extraction, DNA/RNA extraction, nucleic acid, mucolytic agentsAbstract
The importance and application of mucus solutions in sample preparation, a crucial step in genetic material extraction. This enables the extraction of both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) to higher purity and quality. Mucolytic agents, including dithiothreitol, N-acetyl-L-cysteine, and proteinase K, have been employed to reduce the viscosity or enhance the homogeneity of highly viscous samples, such as sputum, saliva, and bronchial lavage fluid. The objective of this study was to collate and present information on commonly used mucolytic agents, including the principles for their appropriate selection for various types of medical samples, to prevent loss and preserve DNA and RNA during nucleic acid extraction. The research methodology involved searching and collecting literature from the PubMed database and other academic sources concerning the use of mucolytic agents in molecular processes, specifically focusing on their impact on nucleic acid extraction efficiency and amplification. The collected literature was analyzed and synthesized to compare key aspects, including properties, mechanisms of action, advantages, limitations, and practical applications of each agent. The findings demonstrated that viscosity-reducing agents significantly enhanced sample homogeneity, reduced false negatives due to sample agglutination, and enhanced the accuracy of biomolecular analysis using the nucleic acid amplification test (NAAT). These agents employed diverse mechanisms, including chemical bond disruption and enzymatic degradation, to optimize samples for the extraction of high-purity DNA and RNA. Crucially, the study highlights that factors such as concentration, exposure time, and temperature must be carefully optimized to prevent inhibition of downstream molecular reactions. Furthermore, integrating these agents with physical methods and rigorous residual removal protocols is essential for significantly increasing the accuracy and efficiency of diagnostic testing.
Downloads
References
Goyani KH, Mukhopadhyaya PN. Evolution of nucleic acid extraction methods and its role in development of
complex human diagnostic solutions with special reference to tuberculosis. Magna Scientia Advanced Research
and Reviews 2023;9(2):115–46.
Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997;63(10):3741-51.
Rubin BK. Mucolytics, expectorants, and mucokinetic medications. Respir Care 2007;52(7):859-65.
Yu F, Qiu T, Zeng Y, Wang Y, Zheng S, Chen X, et al. Comparative evaluation of three preprocessing methods
for extraction and detection of influenza a virus nucleic acids from sputum. Front Med (Lausanne) 2018;5:56.
Bencini MA, van den Brule AJ, Claas EC, Hermans MH, Melchers WJ, Noordhoek GT, et al. Multicenter comparison
of molecular methods for detection of Legionella spp. in sputum samples. J Clin Microbiol 2007;45(10):3390-2.
Deneer HG, Knight I. Inhibition of the polymerase chain reaction by mucolytic agents. Clinical Chemistry
;40(1):171-2.
Liu Y, Kumblathan T, Tao J, Xu J, Feng W, Xiao H, et al. Recent advances in RNA sample preparation techniques
for the detection of SARS-CoV-2 in saliva and gargle. Trends Analyt Chem 2023;165:117107.
Perez-Vilar J, Hill RL. The structure and assembly of secreted mucins. Journal of Biological Chemistry 1999;
(45):31751-4.
Gupta R, Wadhwa R. Mucolytic medications. [Internet]. 2024 [cited 2025 Nov 22].
Available from: https://www.ncbi.nlm.nih.gov/sites/books/NBK559163/
Lukesh JC, Palte MJ, Raines RT. A potent, versatile disulfide-reducing agent from aspartic acid. J Am Chem Soc
;134(9):4057-9.
Hodson ME. Aerosolized dornase alfa (rhDNase) for therapy of cystic fibrosis. Am J Respir Crit Care Med
;151(3 Pt 2):S70-4.
Aldini G, Altomare A, Baron G, Vistoli G, Carini M, Borsani L, et al. N-acetyl-L-cysteine as an antioxidant and disulphide
breaking agent: the reasons why. Free Radical Research 2018;52(7):751-62.
Hill DB, Button B, Rubinstein M, Boucher RC. Physiology and pathophysiology of human airway mucus.
Physiol Rev 2022;102(4):1757-836.
Witten J, Samad T, Ribbeck K. Molecular characterization of mucus binding. Biomacromolecules
;20(4):1505-13.
Helland R, Larsen AN, Smalås AO, Willassen NP. The 1.8 Å crystal structure of a proteinase K-like enzyme
from a psychrotroph Serratia species. The FEBS Journal 2006;273(1):61-71.
Guéroult M, Picot D, Abi-Ghanem J, Hartmann B, Baaden M. How cations can assist DNase I in DNA
binding and hydrolysis. PLoS Comput Biol 2010;6(11):e1001000.
Ye X, Zhang S, Tang M. Effect of sodium citrate anticoagulant solution in continuous veno-venous hemofiltration
in critically ill children. BMC Pediatr 2025;25(1):496.
National Center for Biotechnology Information. Thioglycolicacid. [Internet]. 2024 [cited 2025 Nov. 22].
Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Thioglycolic-Acid
Yu FX, Lin SC, Morrison-Bogorad M, Atkinson MA,Yin HL. Thymosin beta 10 and thymosin beta 4 are both
actin monomer sequestering proteins. J Biol Chem 1993;268(1):502-9.
ตรีวัฒน์วัฒนะโชคชัย, กิ่งกาญจน์ รักษ์มณี. การย่อยสลายเซลล์โฮสต์และอนุภาคไวรัสในการสกัดกรดนิวคลีอิกของไวรัสจากตัวอย่างมนุษย์.
วารสารกรมวิทยาศาสตร์การแพทย์ 2568;67(3):541-56.
Faverio P, Rebora P, Rossi E, Del Giudice S, Montanelli F, Garzillo L, et al. Impact of N-acetyl-L-cysteine on SARS-CoV-2
pneumonia and its sequelae: results from a large cohort study. ERJ Open Res 2022;8(1).
Frazer Z, Yoo C, Sroya M, Bellora C, DeWitt BL,Sanchez I, et al. Effect of different proteinase K digest protocols and
deparaffinization methods on yield and integrity of DNA extracted from formalin-fixed, paraffin-embedded tissue.
J Histochem Cytochem 2020;68(3):171-84.
Brisco MJ, Morley AA. Quantification of RNA integrity and its use for measurement of transcript number. Nucleic Acids
Res 2012;40(18):e144.
Jue E, Witters D, Ismagilov RF. Two-phase wash to solve the ubiquitous contaminant-carryover problem
in commercial nucleic-acid extraction kits. Sci Rep 2020;10(1):1940.
Fischer M, Renevey N, Thür B, Hoffmann D, Beer M, Hoffmann B. Efficacy assessment of nucleic acid
decontamination reagents used in molecular diagnostic laboratories. PLOS One 2016;11(7):e0159274.
Qamar W, Khan MR, Arafah A. Optimization of conditions to extract high quality DNA for PCR analysis from
whole blood using SDS-proteinase K method. Saudi J Biol Sci 2017;24(7):1465-9.
Chang Y, Li T, Niu Y, Guan X, Xie Y. Comparative evaluation of proteinase K and dithiothreitol as pretreatments
for extracting nucleic acids from respiratory samples for multiplex PCR. BMC Microbiol 2025;25(1):233.
Sung H, Yong D, Ki CS, Kim JS, Seong MW, Lee H,et al. Comparative evaluation of three homogenization
methods for isolating Middle East respiratory syndrome coronavirus nucleic acids from sputum samples for
real-time reverse transcription PCR. Ann Lab Med 2016;36(5):457-62.
Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal.
J Appl Microbiol 2012;113(5):1014-26.
Huggett JF, Novak T, Garson JA, Green C, Morris-JonesSD, Miller RF, et al. Differential susceptibility of PCR
reactions to inhibitors: an important and unrecognised phenomenon. BMC Res Notes 2008;1:70.
Boom R, Sol CJ, Salimans MM, Jansen CL,Wertheim-van Dillen PM, van der Noordaa J. Rapid and
simple method for purification of nucleic acids. J Clin Microbiol 1990;28(3):495-503.
Qiagen. QIAamp Viral RNA mini handbook [Internet].2020 [cited 2025 Nov 22].
Available from: https://www.qiagen.com/us/resources/resourcedetail?id=c80685c0-4103-49ea-aa72-8989420e3018&
lang=en
Downloads
Published
How to Cite
Issue
Section
License
Copyright (c) 2026 Ministry of Public Health
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
