A fast and highly efficient automated DNA extraction method from small quantities of bone powder from aged bone samples

Zupanič-Pajnič, Irena (author), Leskovar, Tamara (author), Zupanc, Tomaž (author), Podovšovnik, Eva (author)

An efficient extraction method is important for obtaining high-quality DNA from degraded aged bone samples. An automated full-demineralization method using the EDTA and DNA Investigator Kit (Qiagen) combined with Qiagen’s biorobots was optimized in our laboratory in the past to extract the DNA from 500 mg of aged bone samples. The purpose of this research was to further improve the method with the aim of reducing the required sample material, shortening the extraction time, and achieving higher throughput. To process extremely small samples, the amount of bone powder was reduced to 75 mg, EDTA was replaced with reagents from the Bone DNA Extraction Kit (Promega), and decalcification was shortened from overnight to 2.5 h. Instead of 50 ml tubes, 2 ml tubes were used, which allows higher throughput. The DNA Investigator Kit (Qiagen) and EZ1 Advanced XL biorobot (Qiagen) was used for DNA purification. A comparison between both extraction methods was made on 29 Second World War bones and 22 archaeological bone samples. The differences between both methods were explored by measuring nuclear DNA yield and STR typing success. After cleaning the samples, 500 mg of bone powder was processed using EDTA, and 75 mg of powder from the same bone was processed using the Bone DNA Extraction Kit (Promega). DNA content and DNA degradation were determined using PowerQuant (Promega), and the PowerPlex ESI 17 Fast System (Promega) was used for STR typing. The results showed that the full-demineralization protocol using 500 mg of bone was efficient for Second World War and archaeological samples, and the partial-demineralization protocol using 75 mg of bone powder was only efficient for the Second World War bones. The improved extraction method—for which significantly lower amounts of bone powder can be used, the extraction process is faster, and higher throughput of bone samples is possible—is applicable for genetic identification of relatively well-preserved aged bone samples in routine forensic analyses.

• Language: English
• Keywords: bone DNA extraction, ancient DNA, missing person identification, Second World War, STR typing
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine; FF - Faculty of Arts
• Publication status: Published
• Publication version: Version of Record
• Year: 2023
• Number of pages: 7 str.
• Numbering: Vol. 65, art. 102882
• PID: 20.500.12556/RUL-148310-1e4c979a-ff1f-1684-3447-8aa9e7810cdf
• UDC: 61
• ISSN on article: 1878-0326
• DOI: 10.1016/j.fsigen.2023.102882
• COBISS.SI-ID: 150737923
• Publication date in RUL: 11.08.2023

Record is a part of a journal

• Title: Forensic science international. Genetics
• Publisher: Elsevier
• ISSN: 1878-0326
• COBISS.SI-ID: 32934361

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: ekstrakcija kostne DNK, starodavna DNK, identifikacija pogrešane osebe

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: J3-3080
• Name: Določitev predniškega porekla iz DNA za identifikacijske namene