Chemical contamination in the extracted DNA affects the results of HLA typing by the PCR-SSOP method.

Autor: Shirizadeh A; Student Research Center, Hamadan University of Medical Sciences, Hamadan, Iran.; Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, P.O. Box: 6517838736, Mahdieh Ave, Lona Park, Hamadan, Iran., Ebrahimpur M; Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, P.O. Box: 6517838736, Mahdieh Ave, Lona Park, Hamadan, Iran., Soltani S; Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Hamadan, Iran. Soltanisomaieh@gmail.com.; Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, P.O. Box: 6517838736, Mahdieh Ave, Lona Park, Hamadan, Iran. Soltanisomaieh@gmail.com., Solgi G; Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, P.O. Box: 6517838736, Mahdieh Ave, Lona Park, Hamadan, Iran. gh.solgi@umsha.ac.ir.; Cancer Research Center, Institute of Cancer, Avicenna Health Research Institute, Hamadan University of Medical Sciences, Hamadan, Iran. gh.solgi@umsha.ac.ir.
Jazyk: angličtina
Zdroj: Molecular biology reports [Mol Biol Rep] 2024 Nov 28; Vol. 52 (1), pp. 23. Date of Electronic Publication: 2024 Nov 28.
DOI: 10.1007/s11033-024-10125-3
Abstrakt: Background: The purity of the extracted DNA is critical for successful molecular testing. This study aimed to compare the effect of various DNA extraction methods, extraction processes, and sources of consumables, such as microtubes, on PCR results.
Methods: DNA extraction from whole blood was performed using four different approaches utilizing four types of microtubes: chloroform-based, sodium perchlorate-based, heat-assisted salting out, and solid-phase extraction. Extracted DNA was evaluated by nanodrop spectrophotometry and used in PCR-based methods for human leukocyte antigens (HLA) typing.
Results: The lowest and highest concentrations of extracted DNA were observed in the column-based and heat-assisted methods, respectively. The mean ratios of A260/A230, represents chemical contamination, were significantly lower in all extraction methods using all types of microtubes versus "A" microtube. We observed the highest mean ratio of A260/A230 (> 1.9) in the sodium perchlorate method by using the "A" microtube compared to other types of microtubes and extraction methods. Extracted DNA samples using microtube "A" showed acceptable results for HLA typing compared to other microtubes that represented uninterpretable results in the PCR using the sequence-specific oligonucleotide probe (PCR-SSOP) method.
Conclusion: Our findings indicate that the chemical contaminations derived mainly from microtubes can decrease the quality of DNA and consequently interfere with the amplification or hybridization reactions in the PCR-SSOP method for HLA typing. Although, technical issues and extraction processes may also influence the quality of DNA.
Competing Interests: Declarations. Ethical approval: This study was approved by our institutional ethics committee (IR.UMSHA.REC.1402.219). Study highlights: What is the current knowledge? Impact of the DNA impurity on amplification in the PCR-based method either as inhibition of PCR or insufficient amplification. What is new here? The effect of chemical contamination derived from microtubes on PCR-SSOP results for HLA-DNA typing. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests.
(© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)
Databáze: MEDLINE
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