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  • Chemical contamination in the extracted DNA may affect high-throughput PCR-based methods
  • Ata Shirizadeh,1 Mozhdeh Ebrahimpur,2 Somaieh Soltani,3 Ghasem Solgi,4,*
    1. Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
    2. Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.
    3. Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
    4. Department of Immunology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran.


  • Introduction: 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 disposables such as microtubes on PCR results.
  • Methods: DNA extraction from whole blood was performed by four different approaches: chloroform-based, sodium perchlorate-based, heat-assisted salting out, and solid phase extraction. Extracted DNA was evaluated by Nanodrop spectrophotometry and used for HLA typing by PCR-based methods.
  • Results: The lowest and highest concentrations of extracted DNA were observed in the column-based and heat-assisted methods, respectively. Maximum A260/A230nm ratios were observed in the sodium perchlorate and chloroform methods using A and D microtubes, respectively. Moreover, significant differences were observed in terms of the A260/A230nm ratio of the extracted DNA in chloroform-based extraction using four different types of microtubes. Analysis of extracted DNA using different microtubes in the same method through PCR-SSP and PCR-SSOP indicated that PCR-SSP was run without any issue with interpretable results, but PCR-SSOP was disturbed using the same samples, and results were uninterpretable.
  • Conclusion: Our results indicate that the chemical contaminations derived mainly from microtubes may decrease the quality of DNA and consequently interfere with the amplification or hybridization reactions in the PCR-SSOP method for HLA typing.
  • Keywords: DNA extraction-microtube-PCR, HLA typing