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Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.

Current methods for pregnancy diagnostics are based on immunodetection of pregnancy-specific proteins and in a forensic context suffer from sensitivity and specificity issues. Here, we applied reverse transcriptase polymerase chain reaction (RT-PCR) technology to 11 genes previously reported with placental mRNA circulating in maternal blood. We found two genes, hPL and βhCG, with pregnancy-specific expression in whole blood samples. RT-PCR detection of hPL was positive in all samples tested throughout the pregnancy, whereas βhCG was detectable until half of the second trimester but not at later gestation ages. For hPL, in vitro stability of the transcript was demonstrated until 2 months of age, and the hPL-specific RT-PCR assay applied was highly sensitive with reliable detection from down to 0.25 cm2 dried bloodstain. We therefore suggest hPL-specific RT-PCR as a new molecular tool for forensic pregnancy diagnostics from dried blood stains. Moreover, our results indicate that the time-wise reverse expression of hPL and βhCG during pregnancy may allow an RT-PCR-based estimation of the gestational age from blood stains, adding to the value of forensic pregnancy diagnosis for crime scene investigations. Electronic supplementary material The online version of this article (doi:10.1007/s00414-008-0315-6) contains supplementary material, which is available to authorized users.

In forensic science, the unequivocal identification of the cellular origin of crime scene samples used for DNA profiling can provide crucial information for crime scene reconstruction. We have previously shown that various mRNA markers from genes with expression patterns specific for blood and saliva can be established from whole-genome expression analysis of time-wise degraded samples and were stable enough to specifically identify blood and saliva stains up to 180 days of age. Here, we showed that nine blood-specific and five saliva-specific mRNA markers can be amplified successfully and reliably in much older blood (13-16 years) and saliva (2-6 years) stains, respectively, suggesting their suitability for tissue identification in forensic case work. Moreover, our findings imply that forensic RNA testing can be reliable and robust if degraded samples are considered in the marker ascertainment procedure, with promising expectations beyond tissue identification purposes.

A series of experiments has been performed to evaluate amplification and typing of the D1S80 VNTR locus. The validation study that has been carried out showed that correct D1S80 typing results can be obtained when a defined amplification protocol and a high-resolution polyacrylamide gel electrophoresis method are used. The use of the Chelex extraction protocol has substantially reduced the processing time. DNA-extraction, amplification and subsequent typing can be performed in one day. The discrimination power of this locus is 0.94 in a Dutch Caucasian population sample. The system is extremely sensitive: 0.1 ng of genomic DNA gave a correct typing result. The test could also detect the correct genotypes in mixed samples containing DNA from different individuals. Even if the major type was in a 20-fold excess, the minority type could still be amplified and typed correctly. We have found no deviation from Hardy-Weinberg equilibrium in a Dutch Caucasian population sample. Evidence for the somatic stability of this locus was obtained from a set of experiments where we compared DNA-profiles from corresponding blood, semen and saliva samples. The results of this study suggest that in the near future analysis of the D1S80 locus by DNA-amplification can be applied in actual forensic case work.

If the DNA profiles of a crime stain and the reference sample from the suspect do not match, the suspect is excluded as the donor of the crime stain. However, in some situations the DNA evidence can suggest that a close relative of the suspect might match the stain, in particular when the reference sample from the suspect and the crime stain share rare alleles. This finding can be important for the authorities. The forensic scientist has to decide whether or not to notify the authorities in these circumstances. To the best of our knowledge there is not yet an objective rule for making this decision. We propose such a decision rule for brothers of the suspect, investigate its performance and address some ethical, legal, and practical aspects. Our calculations can be simply adjusted for other relatives of the suspect.

To introduce a duplex PCR system consisting of the STR loci D21S11 and HUMFIBRA in forensic identity testing we analysed a Dutch Caucasian database of 205 individuals. The combined power of discrimination of the two loci is 0.9978 and there was no evidence for linkage equilibrium between the two loci (p = 0.91). However, we noticed departure from Hardy-Weinberg equilibrium for the D21S11-locus in our database (p = 0.03), but the differences between observed and expected D21S11 allele pair frequencies were of negligible practical significance in forensic calculations.

We report on a Dutch population study of the STR loci HUMTHO1, HUMFES/FPS, HUMVWA31/1, and HUMF13A1, in which we used multiplex amplification and automated fragment detection. Genotype and allele frequencies showed no deviation from Hardy-Weinberg and linkage equilibrium. The improved Bonferroni procedure was used to combine the results of several tests. The power of discrimination of a complete profile exceeded 0.9998. We compared the allele frequencies in the Dutch sample to the frequencies in other populations using a bipilot to visualize alleles and populations simultaneously. The Dutch sample was similar to most other Caucasian samples. The data demonstrate that the genetic systems in this report are a valuable tool for forensic identity testing in The Netherlands.

To introduce the loci LDLR, GYPA, HBGG, D7S8, and GC (PM loci) in Dutch forensic identity testing, allele and genotype frequencies were determined in a Dutch Caucasian population sample, which had previously been typed for the HLADQA1 locus [12]. All 6 loci met Hardy-Weinberg equilibrium expectations, and there is little evidence for association between pairs of loci. The combined power of discrimination for all 6 loci is 0.9997. The allele frequencies of the PM loci were similar to 2 other Caucasian populations [3, 10], and differed from 3 non-Caucasian populations [3].

The HLA DQ alpha amplification and typing kit has been designed to be used by the forensic community for purposes of identity testing. The introduction of any new DNA marker in forensic identity testing requires the establishment of a population database for the relevant population(s). To this end allele and genotype frequencies for the HLA DQ alpha locus were determined in a Dutch Caucasian population sample and compared with 7 other population genetic studies. In our population sample the HLA DQ alpha genotype frequencies did not deviate from Hardy-Weinberg expectations and for this locus the power of discrimination is 0.94. A test for homogeneity of the HLA DQ alpha population data based on the allele frequency counts for 8 Caucasian population samples was performed and significant differences were found (P = 0.007). The differences in the frequency of the HLA DQ alpha 2 and 3 alleles are the major cause of this deviation. No deviation from population homogeneity was observed when we compared the genotype frequency distributions among the 8 Caucasian population samples. Combined with the extensive validation studies from Comey and Budowle and Helmuth et al. this population genetic study will allow HLA DQ alpha typing to be used in forensic identity testing in the Netherlands.