Performance of the Human Quantifiler, the Investigator Quantiplex and the Investigator ESSplex Plus kit for quantification and nuclear DNA typing of old skeletal remains

Abstract: Aim. We tested the performance of two real-time polymerase chain reaction (PCR) human quantification kits (Human Quantifiler (Applied Biosystems) and Investigator Quantiplex kit (Qiagen)) and forensic identification short tandem repeat (STR) Investigator ESSplex Plus (Qiagen) kit on approximately 70 years old skeletal remains. Methods. We analysed 54 bones and teeth excavated from Second World War mass graves in Slovenia. Genomic DNA was obtained from 0.5 g of bone or tooth powder after total demineralization. The DNA was purified in a Biorobot EZ1 (Qiagen) device. The same extract was used for quantification and STR typing with all three kits using the amplification conditions recommended by the manufacturers. Results. In almost two thirds of the samples the results of quantification were up to 6 times higher using the Human Quantifiler than using the Investigator Quantiplex kit, because of the degradation of DNA in old skeletal remains and amplification of shorter DNA fragment with the Human Quantifiler kit. The autosomal STR typing with the Investigator ESSplex Plus kit was successful in 52 out of the 54 samples, which represent a 96% success rate. Conclusion. The commercially available Investigator ESSplex Plus kit can be used for STR typing of old skeletal remains with the DNA extraction method optimised in our laboratory and without any changes to the manufacturers' PCR amplification protocols. The Human Quantifiler kit and Investigator Quantiplex kit together can be used for estimation of the degree of DNA degradation in compromised old bone samples. n skeletonised human remains bones and teeth are the only accessible source of DNA which can be preserved for a long time. In them binding of DNA to hydroxyapatite provides stability of DNA and its preservation In addition, the chemistry and methods used for DNA extraction and amplification may have a strong effect on the amplification succes

PKD1 and PKD2 mutations in Slovenian families with autosomal dominant polycystic kidney disease

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder caused by mutations in at least two different loci. Prior to performing mutation screening, if DNA samples of sufficient number of family members are available, it is worthwhile to assign the gene involved in disease progression by the genetic linkage analysis. METHODS: We collected samples from 36 Slovene ADPKD families and performed linkage analysis in 16 of them. Linkage was assessed by the use of microsatellite polymorphic markers, four in the case of PKD1 (KG8, AC2.5, CW3 and CW2) and five for PKD2 (D4S1534, D4S2929, D4S1542, D4S1563 and D4S423). Partial PKD1 mutation screening was undertaken by analysing exons 23 and 31–46 and PKD2 . RESULTS: Lod scores indicated linkage to PKD1 in six families and to PKD2 in two families. One family was linked to none and in seven families linkage to both genes was possible. Partial PKD1 mutation screening was performed in 33 patients (including 20 patients from the families where linkage analysis could not be performed). We analysed PKD2 in 2 patients where lod scores indicated linkage to PKD2 and in 7 families where linkage to both genes was possible. We detected six mutations and eight polymorphisms in PKD1 and one mutation and three polymorphisms in PKD2. CONCLUSION: In our study group of ADPKD patients we detected seven mutations: three frameshift, one missense, two nonsense and one putative splicing mutation. Three have been described previously and 4 are novel. Three newly described framesfift mutations in PKD1 seem to be associated with more severe clinical course of ADPKD. Previously described nonsense mutation in PKD2 seems to be associated with cysts in liver and milder clinical course

Abstracts

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