DNA EXTRACTION FROM POST-MORTEM SAMPLES WITH DIFFERENT DEGREES OF DEGRADATION AND THEIR SUITABILITY FOR ION S5™ NEXT-GENERATION SEQUENCING SYSTEM

Authors

  • Viktorija Belakaposka Srpanova Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Sasho Risteski Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Natasha Bitoljanu Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Ljupcho Chakar Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Ana Ivcheva Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Goran Pavlovski Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Aleksandar Stankov Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Svetlana Krstevska Balkanov University Clinic for Hematology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia
  • Zlatko Jakovski Institute of Forensic Medicine, Criminalistics and Medical Deontology, Faculty of Medicine, Ss. Cyril and Methodius University, Skopje, Republic of North Macedonia

Keywords:

postmortem DNA extraction, DNA quantification, quality control, Ion S5 ™ next-generation sequencing system.

Abstract

Molecular analyses occupy a significant part of laboratory tests in forensic practice. Pre-prepared commercial kits used for DNA extraction are a great choice for samples provided by living people, but in forensic practice most of the samples that undergo molecular processing are often degraded and may pose a challenge in the laboratory. A special challenge is to provide quality genetic material from postmortem samples that would be suitable for further analysis with massively parallel sequencing.

The aim of this study was to introduce and optimize a method for extracting DNA from postmortem specimens with varying degrees of degradation, such as blood, FFPE, and frozen tissue, suitable for Ion S5 ™ sequencing system.

Extraction protocols were modified to increase the quantities and the total yield of DNA. Thus, we doubled the quantity of the analyzed sample, the quantity of buffers and lytic material, and we also extended the incubation time with elution buffer. Quantification was made using Qubit 3.0 fluorimeter, followed by PCR quantification.

Blood produced the best yield of DNA, followed by formalin-fixed paraffin embedded tissue. The type of the sample, the degree of post-mortem damage, as well as the storage time of the sample significantly affect the amount of DNA material as well as its suitability for further analysis. Blood remains the first choice of sample that is suitable for further analysis with the Ion S5 ™ next-generation sequencing system.

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Published

2023-06-16 — Updated on 2023-07-06

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Vol. 3 No. 2 (2023): Acad Med J

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Original Articles