title: DNA quality and quantity from up to 16 years old post-mortem blood stored on FTA cards.
abstract: Blood samples preserved on FTA cards offer unique opportunities for genetic research. DNA recovered from these cards should be stable for long periods of time. However, it is not well established as how well the DNA stored on FTA card for substantial time periods meets the demands of forensic or genomic DNA analyses and especially so for from post-mortem (PM) samples in which the quality can vary upon initial collection. The aim of this study was to evaluate the time-dependent degradation on DNA quality and quantity extracted from up to 16 years old post-mortem bloodstained FTA cards. Four random FTA samples from eight time points spanning 1998 to 2013 (n=32) were collected and extracted in triplicate. The quantity and quality of the extracted DNA samples were determined with Quantifiler(®) Human Plus (HP) Quantification kit. Internal sample and sample-to-sample variation were evaluated by comparing recovered DNA yields. The DNA from the triplicate samplings were subsequently combined and normalized for further analysis. The practical effect of degradation on DNA quality was evaluated from normalized samples both with forensic and pharmacogenetic target markers. Our results suggest that (1) a PM change, e.g. blood clotting prior to sampling, affects the recovered DNA yield, creating both internal and sample-to-sample variation; (2) a negative correlation between the FTA card storage time and DNA quantity (r=-0.836 at the 0.01 level) was observed; (3) a positive correlation (r=0.738 at the level 0.01) was found between FTA card storage time and degradation levels. However, no inhibition was observed with the method used. The effect of degradation was manifested clearly with functional applications. Although complete STR-profiles were obtained for all samples, there was evidence of degradation manifested as decreased peak heights in the larger-sized amplicons. Lower amplification success was notable with the large 5.1 kb CYP2D6 gene fragment which strongly supports degradation of the stored samples. According to our results, DNA stored on FTA cards is rather stable over a long time period. DNA extracted from this storage medium can be used as human identification purposes as the method used is sufficiently sensitive and amplicon sizes tend to be <400 bp. However, DNA integrity was affected during storage. This effect should be taken into account depending on the intended application especially if high quality DNA and long PCR amplicons are required.
authors.affiliation: Laboratory of Forensic Biology, Department of Forensic Medicine, University of Helsinki, P.O. Box 40 (Kytösuontie 11) FI-00014, Finland. Electronic address: anna-liina.rahikainen@helsinki.fi.
authors.forename: Anna-Liina
authors.initials: AL
authors.lastname: Rahikainen
authors.affiliation: Laboratory of Forensic Biology, Department of Forensic Medicine, University of Helsinki, P.O. Box 40 (Kytösuontie 11) FI-00014, Finland.
authors.forename: Jukka U
authors.initials: JU
authors.lastname: Palo
authors.affiliation: Thermo Fisher Scientific, Stockholm, Sweden.
authors.forename: Wiljo
authors.initials: W
authors.lastname: de Leeuw
authors.affiliation: Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX, USA; Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: Bruce
authors.initials: B
authors.lastname: Budowle
authors.affiliation: Laboratory of Forensic Biology, Department of Forensic Medicine, University of Helsinki, P.O. Box 40 (Kytösuontie 11) FI-00014, Finland. Electronic address: antti.sajantila@helsinki.fi.
authors.forename: Antti
authors.initials: A
authors.lastname: Sajantila
mesh_terms: D001801:Blood Stains; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics; D004247:DNA / Q000032:analysis*; D053836:DNA Degradation, Necrotic; D016172:DNA Fingerprinting; D006801:Humans; D018895:Microsatellite Repeats; D013048:Specimen Handling / Q000295:instrumentation*; D013997:Time Factors
chemical_list: D004247:DNA; D019389:Cytochrome P-450 CYP2D6
keywords: DNA extraction; Degradation; FTA cards; Forensic genetics; Inhibition; Quantification