P-215

Polymorphism of four X-chromosomal STRs in a religious minority of Old Believers residing in northeastern Poland

Pepinski W ¹, Niemcunowicz-Janica A ¹, Skawronska M ¹, Janica JR ²,  Koc-Zorawska E ¹, Janica J ¹, Soltyszewski I ³

¹ Department of Forensic Medicine, Medical University of Bialystok, Poland

² Department of Radiology, Medical University of Bialystok, Poland

³ Central Forensic Laboratory of Police, Warsaw, Poland

Old Believers are a fraction of the Russian Orthodox Church who came into existence as a result of schism introduced in 1653-1666 by Patriarch Nikon in opposition to the Russian Church Reform, adopting the liturgy and practices of the Greek Church. The Old Believers who resisted the Reform were condemned and declared dissidents, in 1667 they were separated from the Russian Orthodox Church and severely persecuted under the tsars sought shelter in the most remote corners of Russian Siberia as well as abroad, including USA, Lithuania and Poland. In the 19th century they moved to Suwalki Region (NE Poland), where they founded several villages and have struggled to maintain their religious identity and traditional ways of life, such as farming and continuing to speak Russian. In an effort to preserve their culture, some parents pressured their children to marry young. Many communities lived in almost complete isolation for centuries. Allele frequencies for four X-chromosomal STR (DX8378, DX7132, HPRTB and DX7423) were determined in a population sample of 140 unrelated males and 70 females by multiplex PCR and subsequent automated fluorescent detection (ABI 310) using a commercially available multiplex PCR kit Mentype Argus X-UL (Biotype, Germany). For each locus, allele frequencies were calculated separately for males and females. Comparison of allele frequencies between males and females was performed by the exact test of a RxC contingency table analysis. Possible divergence from Hardy-Weinberg equilibrium (HWE) was tested using the exact test based on 3200 shuffling experiments using the GDA software v1.2. The following statistical parameters were calculated: observed and expected heterozygosity (Ho, He), polymorphism information content (PIC), mean exclusion chance (MEC), expected probability of exclusion (PE) and discrimination power in males (DPM) and in females (DPF). The genotype distributions among the females conformed with Hardy-Weinberg equilibrium except for DX7423 (P=0.0013). A pairwise comparison using the exact test disequilibrium analysis yielded no indication of allelic dependence (0.2438<P<0.6981). No significant differences were observed between allele distributions in males and females (0.4230<P<0.9940), therefore the two groups were pooled into single frequency distributions for respective loci. Kinship tests revealed a typical X-linked inheritance with no mutation. For the quadruplex evaluated, the combined MEC is 0.9919, and the combined DP values are 0.9954 and 0.9999 (for males and females, respectively). A pairwise testing for heterogeneity using the RxC contingency table exact tests for population differentiation according to Carmody revealed significant differences between the group of Old Believers and the autochthonous Polish population at HPRTB and DX7132 (P=0.0010 and P=0.0110, respectively).

Contact: pepinski@amb.edu.pl 

P-216

Y-chromosome variation in northeastern Poland

Pepinski W ¹, Niemcunowicz-Janica A ¹, Skawronska M ¹, Janica JR ²,  Koc-Zorawska E ¹, Janica J ¹, Soltyszewski I ³

¹ Department of Forensic Medicine, Medical University of Bialystok, Poland

² Department of Radiology, Medical University of Bialystok, Poland

³ Central Forensic Laboratory of Police, Warsaw, Poland

Ethnically, Poland has a largely homogeneous population, its percentage of national or ethnic minorities being one of the lowest in Europe, officially estimated at between 3-4% of the inhabitants, which is equivalent to about 1.5 million people. Podlasie in northeastern part of Poland is a frontier region where the influences of various countries and cultures have been clashing for centuries. The region differs from the others due to its scanty population (1.2 million) and ethnical and cultural diversification. It is estimated, that northeastern corner of Poland is inhabited by 200,000-300,000 Belorussians, 20,000-30,000 Lithuanians and also 2,500 Polish Tatars and 600 Old Believers. Lithuanians compose one of the most emancipated, best organized and least assimilated minority communities in the country. Tatars who presently live in Poland are Sunni Muslims. Old Believers are a fraction of the Russian Orthodox Church who came into existence as a result of schism introduced in 1653-1666 by Patriarch Nikon in opposition to the Russian Church Reform. DNA was extracted using the Chelex 100 and proteinase K protocol. The quantity of recovered DNA was determined spectrophotometrically. DNA was amplified in PCR System 9700 (Applera) using commercial kits: PowerPlex Y System (Promega) or genRES DYSplex-1 and genRES DYSplex-2 (Serac). The SWGDAM recommended Y-STR minimal haplotype was considered. Electrophoresis and typing were performed in the ABI 310 Genetic Analyzer (Applera). Reference ladders included in the kits were used for genotype classification. The nomenclature according to the Y-STR Haplotype Reference Database (http://www.yhrd.org) was used. Allele frequencies for each locus were calculated by simple gene counting method. Gene or haplotype diversity/discrimination (GD) and discrimination capacity (DC) values were calculated. AMOVA was performed using the Monte-Carlo test included in the Arlequin software ver. 2.000. The combined values of GD were 0.9836, 0.9750, 0.9815, 0.9638 and 0.9938 for Poles, Belorussians, Lithuanians, Polish Tatars and Old Believers, respectively with corresponding values of DC=0.84, 0.86, 0.82, 0.81 and 0.79 respectively. The pairwise population comparisons between autochthonous Poles and the studied minorities revealed statistically significant differences (P=0.0180, 0.0360, 0.0090 and 0.0000, respectively) and relatively small values of interpopulation variation (R_ST=0.0064, 0.0162, 0.0127 and 0.0311, respectively) indicating a certain degree of genetic differentiation. The resulting data are consistent with the idea of a genetic proximity of Belorussians and Lithuanians to the Polish population due to the common Slavic origin and historical-political contacts. xxx and support the concept of a Polish admixture in Y-chromosomal lineages of Polish Tatars. Old Believers appeared to be more distant from autochthonous Poles which may reflect their different history, religious affiliation and long-established principles of living. We suggest that the differences in some haplotype frequencies should be taken into consideration in certain trace-donor match analyses within the population of northeastern Poland.

Contact: pepinski@amb.edu.pl 

P-217

Sampling efficiency for Amerindian female lineages

Pereira L¹, Goios A^1,2, Amorim A^1,2

¹IPATIMUP (Instituto de Patologia e Imunologia Molecular da Universidade do Porto), Porto, Portugal; ²Faculdade de Ciências da Universidade do Porto, Porto, Portugal

lpereira@ipatimup.pt

Characterisation of mtDNA lineages in South American urban populations has revealed that as much as one third of its gene pool is of Amerindian ancestry, showing the introgression of native American females in the highly mixed (with Eurasian and sub-Saharan) new cosmopolitan communities. By opposition, only a small fraction of the male pool of urban South American populations is of Amerindian ancestry (almost 98% is of Eurasian background). The old native communities were drastically reduced in its effective size, and the remaining few are small and scattered. We could then hypothesize the following scenarios: the native female lineages picked up for the constitution of the new cosmopolitan mixed populations were from communities around the location of the new town and probably still diverse; while the native female lineages observed in present small native communities went through severe demographic forces, such us bottlenecks, genetic drift and founding events. Being these scenarios true, a sampling effort would reveal much more diversity when surveying cosmopolitan populations than small tribes, leading to a reconstruction of the Amerindian mtDNA phylogeny much more promising in the first case than in the second. We compiled published data for South and Central Amerindians and compared their diversity with the cosmopolitan pool of Amerindian ancestry, in order to check if the above described scenarios are discerned in the data: - for native populations: 120 Cayapa from Equator [1]; 129 Yanomami from Venezuela and Brazil [2]; 39 Mapuche from Argentina [3]; 34 Mapuche, 24 Pehuenche and 15 Yaghan from Chile [4]; 46 Ngobe from Panama [5]; 27 Huetar from Costa Rica [6]; 44 Emberá and 31 Wounan from Panama [7]; 22 Arequipa, 61 Tayacaja and 22 San Martin de Pangoa from Peru [8];- for cosmopolitan populations: 44 from Chile and 20 from Colombia [9]; and 82 from Brazil [10]. The Amerindian haplogroup (hap) distribution in the pooled tribal samples was equivalent to the one in the pooled cosmopolitan samples, but the diversity was higher in towns. Haps A and B where the main contributors for the higher diversities observed in towns, while for haps C and D, diversities were almost equal in tribes and towns. This trend of higher hap A and B diversities in towns (as well as the equivalent diversity for haps C and D) was also observed when randomly resampling 4 sub-groups (the same size of towns) inside the pooled tribal sample, showing that this effect is not due to a bias resulting from disproportionate sample sizes. Network analyses showed that in the pooled tribes, haps B and C present a star-like phylogeny, while A and D show several equally frequent haplotypes, being one-step departed in A but many steps in D (leading to a high mean pairwise difference). This testifies the diverse effects acting upon different haps. In pooled towns, networks show a much diverse phylogeny for all haps, as resulting from the picking up of divergent lineages. In conclusion, a considerable amount of information for the native Amerindian lineages can be inferred by studying the descendents of the newly constituted populations after the arriving of Eurasians. This is true for the mtDNA, but, unfortunately, cannot be applied to the Y-chromosome, for which many lineages are lost forever.

 [1] Rickards et al. (1999)     [2] Merriwether et al. (2000) [3] Ginther et al. (1993)        [4] Moraga et al. (2000)        [5] Kolman et al. (1995)             [6] Santos et al. (1994) [7] Kolman et al. (1997)               [8] Fuselli et al. (2003)         [9] Horai et al. (1993) [10] Alves-Silva et al. (2000)

P-218

The Islamization of Iberian Peninsula: a demographic shift or a cultural change? Search for an answer using extant and ancient DNA from Mértola (Southeast Portugal)

Pereira L1, Morales AC2, Goios A1,3, Duarte R1, Rodrigues C2, Endicott P4, Alonso A5, Martín P5, Torres C2, Amorim A1,3

1IPATIMUP (Instituto de Patologia e Imunologia Molecular da Universidade do Porto), Porto, Portugal; 2Campo Arqueológico de Mértola, Portugal; 3Faculdade de Ciências da Universidade do Porto, Porto, Portugal; 4Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford, UK; 5Instituto Nacional de Toxicologia y Ciencias Forenses, Servicio de Biologia, Madrid, Spain

A classical view of the Iberian Peninsula history used to correlate the Islamization of the Iberian Peninsula with significant demographic migrations from North Africa, but, more recently, acculturation phenomena were advanced as being more significant in places like Mértola, an important roman and medieval fluvial port in southeastern Portugal. The Islamization phenomenon has been intensively studied there since the 1980’s. Archaeological excavations lead to the discovery of the “Rossio do Carmo”, a funerary area outside the city walls, where Islamic burials overlap Paleo-Christian ones. A pacific conversion of the inhabitants of Mértola after the Islamic conquest may explain this continuity of use of the early medieval place of burial, better than a massive introgression of North African people. In order to evaluate which of the scenarios fits better the available data, we followed two lines of research on both: (a) from bones recovered from this necropolis; and (b) in the extant population of Mértola.With respect to the ancient mtDNA, three Paleo-Christian individuals (right second metartarsal; 3 upper second premolars and 1 lower second premolar) and one Islamic (right first metartasal and deciduous lower left canine and first molar) were analysed. These samples span a time range from 5th-13th centuries A.D. The amplification of mtDNA, in all the appropriate conditions for the study of ancient DNA, resulted unsuccessful for fragments longer than 123 bp (quantities of DNA templates were minimal), what pointed to degraded sequences, and the presence of multiple sequences in independent PCRs (some of the results could be real, but it wasn't possible to infer which sequences were endogenous). This last fact could be due to 3 plausible causes: post-excavation contamination (excluded by absence of matching between spurious sequences and the ones obtained from the survey of the archaeological team); damaged DNA, causing jumps between templates and generating novel sequences (not cleared up by cloning); and contamination during the burial period, presumably by percolation. This last explanation looks the most probable cause for no reliable DNA sequences being achieved from the Paleo-Christian and Islamic cemeteries of Mértola. So, we were left with the results from the extant Mértola district population. We sampled 43 individuals from the town and from three small villages (Alcaria Ruiva, ancient pre-Islamic foundation; Alcaria dos Javazes, Islamic foundation; and Santana de Cambas, Christian post-Islamic foundation). MtDNA survey revealed that its feminine genetic composition is significantly different from North and Central Portugal, and even from the South of the country. North African lineages are more frequent in Mértola (11.6%) than in any other region of the Iberian Peninsula (the highest is 5% in North Portugal), and the sub-Saharan ones are scarcer than in Southern Portugal (7% comparatively to 11%), while Near/Middle Eastern lineages are much more common (37.2% relatively to 10.9% in Portugal). We are now enlarging the sample in order to obtain a sufficient number of Y-chromosome lineages. Thus, the female lineages from the extant population of Mértola bear a higher proportion of typical components of the Southern and Eastern Mediterranean when compared to other regions of Portugal. Unfortunately, we cannot safely conclude on the time scale for the arrival of these typical southern Mediterranean lineages in the Mértola gene pool. lpereira@ipatimup.pt

P-219

Seventeen Y-chromosome specific short tandem repeat haplotypes study

in Brazilian populations

Pereira RW¹, Hirschfeld GC², Wang AY² and Grattapaglia, D^1,3

¹Graduate Program in Genomic Science and Biotechnology, Catholic University of Brasilia,

²Applied Biosystems, Brasil,

³Heréditas Tecnologia em Análise de DNA Ltda.

The Y-chromosome haplotypes based on high polymorphic STRs are broadly used in forensic laboratories, mainly applied in case of rapes where they drop the number of profile contributors just to male/males without differential DNA extraction necessity. The ultimate commercial set of Y-STRs available to forensic community allows the amplification of 17 loci in a multiplex fashion. This set amplifies loci from the European minimal haplotype, loci from the SWGDAM-recommended Y-STR panel and six additional highly polymorphic loci. The AmpFSTR^â Yfilerä kit has showed in populations studied so far a power of individual discrimination greater than 95%. Here we show the haplotype diversity and some population genetics parameters found with the AmpFSTR^â Yfilerä kit in 274 males from the five geopolitical Brazilian regions. The 274 samples were distributed in 77 samples from the North, 49 samples from the Central west, 49 samples from the Northeast, 36 samples from the Southeast and 63 samples from the South. All males were resident in such regions when they submitted themselves to paternity investigation. The DNA samples were amplified and the PCR products analyzed in the ABI Prism 3100 according to the manufacturer’s protocol. The ABI Prism 3100 sample files were analyzed using the Genescan and Genotyper softwares and once the table with individual STRs genotypes was generated, it was used to create Arlequin package input data file. The Arlequin package was used to automatically investigate haplotype diversity, haplotypes uniqueness and the molecular variation partition among regional groups and within them. The global sample analysis showed 258 unique haplotypes out of the 274 Brazilian Y chromosomes sampled (94.42 % of individual discrimination power). The number of unique haplotypes / total chromosomes for each geopolitical region was 74/77 to North, 48/49 to Central west, 46/49 Northeast, 34/36 Southeast and 62/63 to the South chromosomes. The haplotype diversity in the global sample was 99.95 % (S.D +/- 0.0004). All geopolitical regions samples showed haplotype diversity greater than 99 %. The analysis of molecular variance showed that 99.72 % of the molecular variation was due variation within each geopolitical region group and that 0.28 % was due variation between them. The results found in this work showed that the AmpFSTR^â Yfilerä kit has a high power of individual discrimination and that there is no Y-chromosome structure in Brazilian population, allowing the use of a unique database of Brazilians chromosomes. As described by others we also found some loci with more than one allele. The multi-allelic pattern occurred frequently at the DYS385 loci, twice at the DYS389II and DYS439 and once at the DYS437. One sample showed two alleles at the DYS389II, DYS439 and DYS437. As pointed out by others authors this multi-allelic pattern frequency must be better understood, as they might be taken as sample mixture. Regarding the three loci double allele pattern, at least two independent authors described it in one sample from Spain and other from Bahia, Brasil. The three loci are located in the AZFa segment and its duplication, followed by STRs mutation must the culprit for the double allele pattern. As forensic community broadly uses these three STRs, the  understanding of duplication uniqueness or recurrence is important to realize the real consequence of multi-allelic pattern in forensic casework. contact: rinaldo@pos.ucb.br

P-220

Microsatellite polymorphisms in two Taiwanese aboriginal groups

A.M. Pérez-Miranda ^{1, 3}, M. A. Alfonso-Sánchez ², R. J. Herrera ¹

1 Molecular Biology and Human Diversity Laboratory, Department of Biological Sciences, Florida International University, Miami, FL 33199, USA

2 Departamento de Genética y Antropología Física, Universidad del País Vasco, Apartado 644, 48080 Bilbao, Spain

3 Anglia DNA Bioservices Limited, Norwich Research Park, Colney Lane NR4 7UH, Norwich, UK

In the attempt to reconstruct the prehistory of Pacific and Indian Ocean populations, Taiwan’s aborigines appear to be of particular interest. Linguistic and archeological evidence indicates that the dispersal of Austronesian speakers throughout the islands of Oceania and Southeast Asia may have originated from Taiwan about 5,000 years ago. In the island of Taiwan, formerly known as Formosa, nine indigenous groups have coexisted (Tsou, Bunun, Paiwan, Rukai, Atayal, Saisiat, Ami, Puyuma and Yami), which are highly homogeneous within each tribe, but diversified among the different tribes probably due to long-term isolation. The aim of the present study was the genetic characterization of two of these tribal groups (Ami and Atayal) based on the short tandem repeats (STRs) sanctioned by CODIS (D3S1358, TH01, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPOX, and FGA). The sample included 108 unrelated healthy individuals: 40 Ami and 68 Atayal. Significant departures from genetic equilibrium were detected at the D8S1179 and TH01 loci in the Ami, which persisted even after applying Bonferroni-type corrections. Gene diversity (GD) values ranged from 0.5377 (TPOX) to 0.8674 (FGA) in Atayal, whereas in the Ami subpopulation GD oscillated between 0.6409 (TPOX) and 0.8764 (D21S11). A notable heterozygosity was observed in both tribal groups, although it was slightly higher in the Ami group (average: 0.7867) than in Atayal (0.7036). The information provided by the STR loci was analyzed using distance-based methods (Neighbor-Joining trees and multidimensional scaling), to assess the genetic relationships of these Taiwanese groups with others Asian populations.

Contact: ana@angliadna.co.uk

P-221

Automation of post-mortem or non-standard reference samples genotyping using FTA

Pesquier B, Taillé A, Garcin G, Frackowiak S, Coiffait P-E

   Laboratoire de Police Scientifique de Marseille – Section Biologie – 13245 Marseilles Cedex 04 – France

In forensic biology, reference samples take a growing place in the analysis pool. Today, in France, the vast majority of these are saliva samples on FTA® paper, which can be easily automated*. However, some of them, like post-mortem samples or buccal swabs and brushes, are not standardised.

In this poster, we describe our work toward automation of the analysis of such non-standard reference samples, using FTA® cards (Indicating FTA Microcard, Whatman).

The first step consists of manual spotting of the samples on the FTA® card. For autopsic blood, 50µL are directly applied. Other post-mortem tissues are strongly rubbed on the paper, and swabs or brushes are moistened before application. All the samples are allowed to dry at least overnight.

The second step consists of the genetic analysis of the samples, according to the high-throughput process we developed for standardised reference samples.

The genotypes of the spotted samples are compared to those obtained from the same original samples typed after non-organic extraction – each sample is tested 6 times, to estimate the reproducibility of the results.

Then, the impact on the genotypes' quality of the amplification of 1, 2 or 3 FTA® punches in the same well is assessed.

Our results show that

1) the quality of the profile from a FTA-spotted sample is positively correlated to the quantity of DNA in the reference extract of the corresponding sample.

2) None of the samples lead systematically to acceptable results. At best, the optimal quality of profile is obtained for 4 of the 6 successive tests.

3) In a few cases, the addition of more than one punch in the same reaction can ameliorate the profiles. However, the improvement does not seem to be correlated to the quantity of DNA in the original samples (maybe due to polymerase inhibition by excess of paper in the PCR mix).

In conclusion, the use of FTA cards for non-standard reference samples can be a convenient alternative for DNA typing as it offers all the advantages of the subsequent automatised treatment. Yet, in some cases, multiple analysis may be necessary to obtain a reliable profile (≥ 5 loci meeting the validation criteria), because of the lack of reproducibility of the FTA technology (as already noted for saliva).

* Delpech and coll, INTERPOL Meeting, Lyon, october 2004 “Use of an automated process for DNA typing of buccal samples to supply the french national database”

Contact: Benjamin.PESQUIER@interieur.gouv.fr, Sylvie.FRACKOWIAK@interieur.gouv.fr 

P-222

MALDI-TOF MS analysis of Y-SNPs in ancient samples

Petkovski E^1,2, Keyser-Tracqui C¹, Hienne R², Ludes B¹

¹EA 3428, Institut de Médecine Légale, Strasbourg, France

²Laboratoire CODGENE, Strasbourg, France

Studying ancient central Asian, Siberian and South American populations with classical markers (nuclear microsatellites and mitochondrial DNA sequence polymorphisms) allowed us to investigate parental relationships among individuals from burial sites revealing funeral practices. Ancient DNA can also provide information on the origins and the history of population from the past. Focussing on biallelic markers which have a lower mutation rate than repeat polymorphism it is possible to address events corresponding to longer periods of time. Working on ancient DNA samples from Mongolia, Siberia, Yakutia and South-America we concentrated on three Y chromosomal SNPs (TAT, M242 and RPS4Y) known to have specific allelic distributions in these populations or to be informative regarding the peopling of America (M242 and RPS4Y).

The TAT-C allele is observed at very high frequencies in Yakuts (Pakendorf et al., 2002). The M242 derived allele occurs in all indigenous American Y chromosomes that do not carry the RPS4Y mutation (Seielstad et al., 2003) and also at a non-negligible frequency in central Asian, Indian and Siberian populations. The M242 mutation is widely distributed in Eurasian populations and it arose after the M45 and M74 mutations but before M3 which is before the first migration into the Americas. The RPS4Y₇₁₁ mutation is restricted to eastern Asia and America (Bergen et al., 1999) raising a Native American founder lineage outside M45 characterized by differentiated STR alleles (Lell et al., 2002).

Facing ancient samples where DNA is strongly degraded and scarce requires the use of technologies which can provide information from only short fragments of intact template. We developed a primer extension and MALDI-TOF MS based triplexed reaction for the investigation of these polymorphisms. This sensitive method, based on an intrinsic property of the oligonucleotides not requiring any product labelling, allows taking particular questions in hand as it can be adapted to the sample and its informativity.

contact: elizabet.petkovski@ulp.u-strasbg.fr 

1. Pakendorf B, Morar B, Tarskaia LA, Kayser M, Soodyall H, Rodewald A, Stoneking M. Y-chromosomal evidence for a strong reduction in male population size of Yakuts. Hum Genet. 2002 Feb;110(2):198-200.

2. Seielstad M, Yuldasheva N, Singh N, Underhill P, Oefner P, Shen P, Wells RS. A novel Y-chromosome variant puts an upper limit on the timing of first entry into the Americas. Am J Hum Genet. 2003 Sep;73(3):700-5.

3. Bergen AW, Wang CY, Tsai J, Jefferson K, Dey C, Smith KD, Park SC, Tsai SJ, Goldman D. An Asian-Native American paternal lineage identified by RPS4Y resequencing and by microsatellite haplotyping. Ann Hum Genet. 1999 Jan;63 ( Pt 1):63-80.

4. Lell JT, Sukernik RI, Starikovskaya YB, Su B, Jin L, Schurr TG, Underhill PA, Wallace DC. The dual origin and Siberian affinities of Native American Y chromosomes. Am J Hum Genet. 2002 Jan;70(1):192-206. Epub 2001 Nov 30.

P-223

Forensic DNA typing of human nails at various stages of decomposition

Piccinini A¹, Cucurachi N², Betti F¹, Capra M¹, Lorenzoni R¹

¹Istituto di Medicina legale. Università degli Studi di Milano

²Dipartimento di Anatomia umana, farmacologia e Scienze medico-forensi. Università degli Studi di Parma

Forensic scientists often face the problem of extracting and typing human DNA from highly degraded materials such muscle and bones from decomposed bodies.

Bone samples are particularly difficult and time consuming to be analysed and other body tissues suffer from rapid deterioration.

Nails are a well-known source of DNA and their composition makes them less predisposed to decomposition compared to other soft tissues.

The aim of this study was to evaluate the usefulness of DNA extracted from aged human nails in forensic cases.

We analysed human nails taken either from exhumed and partially skeletonised bodies or from nail clippings stored at room temperature for more than 10 years.

DNA was extracted with phenol-chlorophorm and typed with STRs using commercial kits.

The adopted DNA extraction procedures yielded enough DNA for reliable PCR results even when no results were obtained either from soft and bone tissues.

This study thus confirms the usefulness of nails as a good source of DNA even in cases when PCR failed to amplify DNA extracted from bones

contact andrea.piccinini@unimi.it

P-224

Y-STR typing in the identification of genetic profile of the semen

Pinheiro MF^1,2, Pereira MJ¹, Cainé L¹, Lima G¹, Pontes L¹, Abrantes D¹

¹ Instituto Nacional de Medicina Legal – Delegação do Porto

²Faculdade de Ciências da Saúde – Universidade Fernando Pessoa

The Genetics and Biology Forensic Laboratory of the National Institute of Legal Medicine (Oporto Delegation) has been asked to solve criminal cases, among other analysis, being the majority of them sexual female assaults. For a variety of reasons, some victims of sexual aggressions provide vaginal samples more than 24-36 h after the incident. In these situations, the ability to obtain an autosomal STR profile of the semen donor reduces as the post-coital interval is extended. Therefore, we have used Y-STR loci to obtain a genetic male haplotype even when the autosomal STRs failled. DNA was extracted from samples collected in sexual female cases using the organic phenol-chloroform-isoamylalcohol method. The loci were co-amplified using the PowerPlex^® 16 System (Promega) or the AmpFℓSTR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) for the autosomal STR systems and, for the Y-STR systems, the PowerPlex^® Y System (Promega) and, in some situations, the AmpFℓSTR^® Yfiler™ (Applied Biosystems). The amplified products were detected and separated by capillary electrophoresis on an ABI PRISM^® 310 Genetic Analyzer (Applied Biosystems). Fragment sizes were determined automatically using the Genescan^® Anlysis Software v 3.7 and allele designations using Genetyper^® Software v. 3.7 (Applied Biosystems) typed by comparison with an allelic ladder. In this study we demonstrate, through some examples, that Y-STR systems provide a complete male haplotype despite the cytological absence of spermatozoa, the negative acid phosphatase reaction and without the male autosomal profile. These evidences show the efficacy and high sensivity of Y-STRs for discerning the genetic profile of the male donor in admixtures of body fluids, mainly when the female component was present in vast excess.

Contact: Biologia@dpinml.mj.pt

P-225

BPA analysis as a useful tool to reconstruct crime dynamics. Part II

Pizzamiglio M¹, Fratini P¹, Floris T¹ , Cappiello P¹, Matassa A¹ , Festuccia N¹ and Garofano L¹

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

This paper concerns a case of a gruesome double murder committed by two minors, a girl and her boyfriend, who killed a 40 year old woman and her son, who was just 12. The victims were the mother and the young brother of the girl and the murder was committed in their house, as the victims came back from the gym.

We refer to technical activities we conducted at the crime scene and the analytical approach we adopted,  based on DNA as well as on BPA analyses of the bloodstains we recovered, studied and collected during CSI.

Following this integrated analytical approach, also supported by fingerprint and footprint exams, it was possible to understand the role of the two young killers and thus reconstruct the dynamic of the event.

lugaro@tin.it

P-226

The use of mini STRs on degraded DNA samples

Pizzamiglio M¹,  Marino A¹,  Coli A¹, Floris T and Garofano L¹.

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

Forensic laboratories, much more frequent than in the recent past, have to face degraded evidence, which usually contains small amounts of DNA (LCN DNA). With these exhibits, even relying on the most efficient extraction system, or amplifying with increased number of PCR cycles, the STRs profiles are often incomplete or exposed to stochastic effects. In this paper we refer to the use of a mini pentaplex (FGA, D21S11, CSF1PO, D7S820 and TH01) and a mini quadruplex (Penta D, D2S1338, Amelogenin and D18S51) used to analyse casework samples which gave negative or very partial results, when analysed by the kits commercially available.

The results we obtained, still preliminary, really encouraged us to continue these studies because evidence, negative to the quantification procedures or exhibiting DNA concentration of 50 picograms or less, gave reliable data.

lugaro@tin.it

P-227

STRs typing of DNA extracted from cigarette butts soaked in flammable liquids for several weeks

Pizzamiglio M¹, Marino A¹, Maugeri G¹ and Garofano L¹

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

This paper refers to a case of arson, in which we analyzed three cigarette butts, apparently smoked, collected from a crime scene when they were still soaked in a petroleum blend used to ignite the fire. DNA extraction was carried out using QIAamp 96 DNA Swab BioRobot kit procedure. The amount of human DNA recovered was then quantified by slot-blot hybridization with the chemiluminescent signals recorded by GeneGnome CCD imaging system, whose values ranged from approximately 0.01 to 0.1 µg/µl. Two complete male profiles based on 17 STRs were obtained from two out of three cigarette butts, while a mixed profile compatible to the previous two individuals was obtained from the third butt. Chemical analyses suggested that the cigarette butts had been left soaking in a mixture of diesel and kerosene oils for at least 45 days before they were collected and sent to the lab.

Due to these situations, we decided to carry out two experimental trials in order to establish the possibility of extracting and successfully typing DNA from cigarette butts, smoked by the same individual, under the same conditions as we faced with the evidence described above. The trials were carried out as follows:

1) The first set of three cigarettes were left soaking in three common flammable liquids (alcohol, gasoline and diesel oil) for 12, 24 and 72 hours ;

2) The second set of three cigarettes were soaked in the same liquids, but for a longer periods time of 1, 2 and 3 months.

 A total of 54 cigarette butts underwent STRs analyses. The results of the two trials were as follows: all samples were successfully typed, showing the great possibility of DNA analysis, even when exhibits are recovered from very critical situations and DNA is present  in very low quantities.

lugaro@tin.it

P-228

The importance of a well defined analytical strategy to solve complex murder cases

Pizzamiglio M¹, Marino A¹, Maugeri G¹, Stabile M¹ and Garofano L¹

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

Forensic techniques are becoming more and more powerful and affordable. This allows labs to utilise precise strategies, permitting multiple analytical approaches on the same evidence, thus obtaining precious information to solve criminal cases.

This paper refers to a murder in which we received a plastic bottle and a latex glove. These items were collected near a stolen car used in  the commission of  the murder, and then burnt in order to destroy evidence linked to the murder.

 Regarding the bottle, we collected samples of saliva from the neck of the bottle.  The glove underwent  three different analyses, which were:

-         sampling and genetic analyses of sweat traces taken from the internal surface of the glove, corresponding to the lower palm area ;

-         development of palm-prints from the internal surface of the glove, corresponding to the upper palm area ; 

-         collection of gun shot residues (GSR) from the edge of the glove.

 Two fully genetic profiles were obtained from the biological traces collected from the glove. The analyses of the glove was instrumental in permitting the identification of the shooter who had played an important role in the murder.

lugaro@tin.it

P-229

Robotic DNA extraction system as a new way to process sweat traces rapidly and efficiently

Pizzamiglio M¹, Marino A¹, My D¹ Bellino C¹ and Garofano L¹

¹ Raggruppamento Carabinieri Investigazioni Scientifiche, Reparto di Parma, Italy

A modified DNA IQ^TM  System (Promega Corporation, USA) was used on a variety of exhibits collected at different crime scenes, potentially interested by sweat traces and analysed by means of a fully automated extraction (Multiprobe II plus EX by ABD, USA).

As is well known, sweat usually soaks large portions of fabrics and clothes. Variable preliminary treatment steps are needed to isolate the few cells eventually still present in each item.

The goal of our application consisted in setting up a dedicated robotic extraction in order to manage large volumes of lysis buffer, to facilitate processing of large portions of evidence.

We started with a melted volume of 10-15 ml, with a recovery of purified DNA between 500 and 2000 picograms, which allowed us to obtain full STR profiles, saving time, improving the rate of success on LCN samples and reducing the risk of possible contamination.

lugaro@tin.it