P-245

Quantification of human DNA by Real Time PCR in forensic casework

Ricci U¹, Marchi C², Previderè C³, Fattorini P⁴

Azienda Ospedaliera-Universitaria  “A.Meyer”, Florence, Italy

¹U.O. Genetica Medica

²Laboratorio di Analisi Chimico Cliniche e Microbiologia²

³Dipartimento di Medicina Legale e Sanità Pubblica, Università di Pavia, Italy

⁴Istituto di Medicina Legale di Trieste, Italy

The estimation of the amount of human DNA is a recommended procedure in forensic casework. We extensively employed a Real-time quantitative PCR system together with a commercially available kit (Quantifiler™ Human DNA Quantification Kit, Applied Biosystems), for the quantification of human DNA in a large variety of samples. Preliminarly, fifty DNA extracts from blood, thirty from bone marrow, ten from fresh bone, twenty from saliva and various swabs from gloves used by a known donor, were dosed with this kit and compared with the spectrophotometric determination at 260 nm. The method was then used to quantify forensic DNA extracts from blood stains, sperm, vaginal swabs, hairs, old bones and teeth and DNA recovered from touched objects. In many cases duplicated analyses were performed to evaluate the reliability of the results. Generally, a low deviation standard for the same sample was observed. However, when the inhibitors in a sample were in high concentration and/or DNA degradation was present (like in post-mortem matrices), we observed variable results of quantification in the same extract. In forensic samples from touched objects or containing low copy number DNA, PCR amplifications with commercial and home-made kits using dosed template DNA with Real-time, were performed. In these samples it is very important to know the DNA concentration to state how many replicate analyses are required for statistically reliable results. Moreover, DNA extracts from vaginal swabs containing sperm and vaginal cells were also dosed with a Y-specific human DNA system (Quantifiler™ Y - Human DNA Quantification Kit, Applied Biosystems), to evaluate the proportion of the male component in these samples.

PCR amplification reactions were prepared from these samples, using AmpFlSTR® Profiler Plus™ (Applied Biosystems) and a Y-specific amplification kit (PowerPlex® Y System, Promega). Here, we discuss the correlation between DNA quantification and PCR amplification in different forensic samples normally recovered in forensic casework.

contact: u.ricci@meyer.it

P-246

Analysis of twelve X-chromosomal short tandem repeats in the Northwest Italian population by means of two multiplex PCRs

Robino C, Giolitti A, Gino S, Torre C

Department of Anatomy, Pharmacology and Legal Medicine, University of Turin, Turin, Italy

The analysis of short tandem repeats (STR) located on the X chromosome can effectively complement autosomal STR data in selected cases of kinship testing: deficiency paternity cases; paternity cases involving close blood relatives as alternative alleged fathers; maternity testing of male children. The use of X-STRs in forensic practice requires a precise knowledge of the population genetics properties of these markers: degree of polymorphism and allelic distribution in different population samples, testing of Hardy-Weinberg equilibrium, possible evidence of linkage disequilibrium. At present, population data on X-STRs available in forensic literature are however rather limited.

In this study, twelve of the most commonly used X-STRs were analysed in a sample of 140 unrelated Italians (70 females and 70 males) residing in Piedmont (Northwest Italy) by means of two multiplex PCRs. Multiplex PCR “I” included markers DXS6789, HumARA, GATA172D05, DXS101, DXS8378, and DXS8377; PCR “II”, loci DXS7132, DXS6800, DXS6803, DXS7424, HPRTB, and DXS10011. Typing was done on ABI PRISM 310 Genetic Analyzer in comparison to sequenced allelic ladders and control DNA (K562 cell line).

Allelic frequencies of the twelve X-STR loci in the Northwest Italian population were determined by direct gene counting: a duplication at the locus DXS6789 was observed in a male individual. All loci were found to be in Hardy-Weinberg equilibrium. No significant differences in the allelic frequencies of female and male samples were observed by exact test. Parameters of forensic interest - heterozygosity, polymorphism information content (PIC), power of discrimination in females (PD^F) and males (PD^M)- were calculated. Heterozigosity of X-STR loci ranged between 0.942 (DXS10011) and 0.686 (DXS8378); PIC between 0.935 (DXS10011) and 0.605 (DXS8378). Locus DXS10011 showed the highest values of PD^F (0.992) and PD^M (0.934), while DXS8378 the lowest:  PD^F (0.832) and PD^M (0.646). Inter-marker linkange disequilibrium was analysed in the male sample: significant linkage disequilibrium (p < 0.01) was observed for the closely linked loci HumARA and DXS7132.

Contact: carlo.robino@unito.it 

P-247

Gene frequencies of six miniSTR in Tuscany (Italy)

Anna Rocchi¹, Isabella Spinetti¹, Chiara Toni¹, Silvano Presciuttini², Ranieri Domenici¹

¹Unit of Legal Medicine, School of Medicine, University of Pisa, Italy

²Center of Statistical Genetics, University of Pisa, Italy

In a recent work [1], six STR loci with amplification products smaller than 150 bp have been characterized using a protocol involving two triplexes. These loci, called “miniSTR”, represent promising tools for recovering genetic information from degraded DNA samples for which the currently used loci generate partial profiles.

Here, we report preliminary population data from a sample of unrelated subjects born in Tuscany. The following table shows the number of alleles detected and the expected heterozygosity.

Marker	N. alleles	Heterozygosity
D10S1248	8	0.794
D14S1434	5	0.777
D22S1045	5	0.785
D1S1677	7	0.841
D2S441	7	0.834
D4S2364	5	0.751

1.      Michael D. Coble, John M. Butler. Characterization of New MiniSTR Loci to Aid Analysis of Degraded DNA. J Forensic Sci. 50 (1):43-53 (2005)

Contact: sprex@biomed.unipi.it 

P-248

Allele frequency of 12 Y-STR loci in the Brazilian population from South Brazil

Rodenbusch R¹, Mardini AC¹, Estivalet AAF¹, Gastaldo AZ¹, Schumacher S¹, Albarus MH¹, Giugliani R^1,2, Saraiva-Pereira ML^1,3.

¹Medical Genetics Service, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil

²Genetics Department, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

³Biochemistry Department, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

Genetic individuality is based on genome variability among individuals. Analysis of short tandem repeats (STR) polymorphic markers is commonly used in paternity testing as well as forensic cases. Although autosomal STRs are commonly used in these cases, STRs in Y-chromosome can provide useful information in paternity investigation cases where alleged father cannot be tested and in other investigation cases where paternal lineage identification can be assessed. The aim of this study was to determine, in the regional population, allele frequency of 12 loci (DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, and DYS439) included in PowerPlex Y kit (Promega) and mutation rate in each loci. Population tested was composed by 162 pairs father/son that paternity was previously tested and confirmed using autosomal STRs, with a minimal probability of 99,99%. DNA isolation was performed from 300μl of each sample using the Wizard^® Genomic DNA Purification Kit (Promega), according to manufacture instructions. Regions of interest were amplified by multiplex-PCR using fluorescent primers, using PowerPlex Y kit (Promega). Amplification products were analyzed in an ABI Prim^Ò 3100 Genetic Analyzer, and GeneScanÒ and GenotyperÒ software. In this sample population, we have found 151 distinct haplotypes and mutation was reported in the following loci: DYS19, DYS390, DYS439, and DSY437. These results allowed the establishment of allele frequencies for the above Y-STRs in our population. These frequencies can be now used in our lab, a reference center for paternity cases, mainly in paternity cases where the alleged father cannot be tested. We were also able to determine the limited mutation rate in our population using this set of markers. Finally, this study allowed the establishment that these STRs show a high discriminating rate in our population, being valuable to be employed in either cases of paternity testing as well as forensic genetic.

contact: mlpereira@hcpa.ufrgs.br

P-249

Evaluation of 12 single-copy and 2 multi-copy Y-chromosomal STR loci in five German populations

Rodig H¹, Grum M¹, Grimmecke H-D¹, Roewer L²

¹Biotype AG, Moritzburger Weg 67, 01109 Dresden, Germany

²Institute of Legal Medicine, Charité - Universitary Medicine, Hannoversche Str. 6, 10115 Berlin, Germany

The Minimal Haplotype (MH) loci, definitely DYS19, DYS385, DYS389-I, DSY389-II, DYS390, DYS391, DYS392, and DYS393 were evaluated by the International Forensic Y-User Group as a standard for Y-chromosomal STR-typing. In the meantime the complete sequence of the Y chromosome was screened for microsatellites and further highly discriminating Y-STR loci were identified [1, 2].

We have developed two multiplex applications for Y-STR typing. One multiplex which is also commercial available is suitable for the analysis of the MH loci, named Mentype^® Argus Y-MH PCR Amplification Kit.

A second multiplex was designed for DYS446, DYS447, DYS448, DYS449, DYS463, and DYS464 typing. These Y-chromosomal STRs were evaluated with regard to gene diversity (D) in 5 German population groups. The resulting data showed DYS464 (D = 0.7733 - 0.9446), DYS449 (D = 0.7844 – 0.8511), and DYS385 (D = 0.7511 – 0.8433) on the top of the newly established ranking list. The need for further loci was shown in one forensic case where the two haplotypes of the piece of evidence and the suspect person could only be discriminated by DYS446.

References

[1] Redd et al., Forensic Sci Int 130:97-111, 2002

[2] Kayser et al., Am J Hum Genet 74:1183-97, 2004

Correspondence to

Dr. rer. nat. Heike Rodig, Biotype AG, Moritzburger Weg 67, 01109 Dresden, Germany

Tel.: +49-351-8838400, Fax: +49-351-8838403

E-Mail: h.rodig@biotype.de

P-250

The male genetic history of the Sorbs – a Slavic island population in Germany

Roewer L¹, Willuweit S¹, Rodig H², Groß A², Weidlich S², Kayser M³, Nagy M¹

¹Institute of Legal Medicine, Charité-University Medicine Berlin, Hannoversche Str. 6, 10115 Berlin, Germany

²Biotype AG, Moritzburger Weg 67, 01109 Dresden, Germany

³Department of Forensic Molecular Biology, Erasmus University Medical Centre Rotterdam

PO Box 1738, NL-3000 DR Rotterdam, The Netherlands

The about 67.000 Sorbs living in the provinces of Upper and Lower Lusatia represent the only autochthonous Slavic-speaking population in Germany. Linguistically the Sorbs belong to the Western Slavs and are closely related to the Poles and Czechs. Whereas the ancient Sorbs living in Poland underwent complete assimilation, they managed to exist as a bilingual minority in Eastern Germany until today.

The Y-chromosome haplotype analysis in a sample of 30 unrelated Sorbian males reveals a significant distance to the German neighbouring populations and a close vicinity to the Polish population samples. Nevertheless, the most frequent Sorbian Y-chromosomal minimal haplotype 17,13,31,25,10,11,13,10-14 typed in roughly 14 % of the analysed 30 Sorbs is relatively rare in Poland (Fig.1, Fig.2).

A closer look reveals a one-step deviation of this Sorbian modal haplotype from that of the Poles (17,13,30,25,10,11,13,10-14; highest frequencies in Wroclaw – 7.4%, Krakow – 6.5 %, Warsaw 5.41 %, Gdansk 4.2 %).

The working hypothesis is to assume that the isolation of the early Sorbian (Surbi) families on the Eastern Franconian territory (colonization of Lusatia by the Franks starts in 1104) and later in Germany and its separation from Poland and the Bohemian/Czech territories led to the observed differentiation. Only half of the Sorbian population survived the Thirty-year-war 1618-48. This bottleneck may further explain the current reduced haplotype diversity. 

The in-depth analysis of the Sorbian modal haplotype and its neighbours by up to 39 Y-STR markers reveals a recent split of the Sorbian-Polish lineages. This fits well with the hypothesis of a separation of the Lusatian Sorbs from the Western Slavic Poles within recent historical time. Contact: lutz.roewer@charite.de

 

Fig. 1.  20 most frequent haplotypes in Sorbs and Poles (Wroclaw)

Fig.2 Distribution of the Sorbian modal haplotype among YHRD population samples

P-251

A novel approach for genotyping of LCN-DNA recovered from highly degraded samples

C. Romano, E. Di Luise, D. Di Martino, I. Ciuna and L .Saravo*

¹ Laboratory of Molecular Biology – Raggruppamento Carabinieri Investigazioni Scientifiche (RaCIS), 98128 Messina – ITALY.

Nowadays Short Tandem Repeats Polymorphisms are the most utilized genetic markers in forensic biology. They have become the markers of choice for human identification protocols applied in case of mass fatality disaster. Unfortunately in the latter event STR-based methods easily result in amplification failures due to the extremely low DNA quality together with the presence of contaminants.

A new kind of STRs profiling system is based on the amplification of shorter fragments compared to the conventional STR multiplexes; because of their length, these “Mini” STR amplicons could be obtained even from extremely degraded DNA and/or from very few copies of template DNA. Mini STR kits for Human Identification have been already validated and placed on the market; moreover they are related to those loci which are commonly detected by traditional STRs kits, allowing comparison between data yielded with both methods. For all the above mentioned reasons, “Mini” STR represents more suitable and robust markers system which could be applied even in case of spoiled completely burnt and anyway degraded DNA samples.

 In this paper we report an homicide casework occurred in the South of Italy: a young guy was killed in a farmhouse and then burnt. Nothing inside could be collected apart a burnt stub which was found totally submerged in mud and wet ash. It was the only sample we could process in short time in order to perform a comparison with the likely family of origin and then with the burnt organic residuals. Hence a comparative genotype analysis between STRs and “Mini” STRs was carried out in order to verify reliability, efficiency and discrimination power of the latter ones.  

Our results show that “Mini” STRs permit a reliable determination of 9 human allelic loci whereas traditional STRs typing protocols offer only a partial resulting genotyping profile heavily reducing the chance of identification.

In our experience “Mini” STRs turned out to be more robust and sensitive markers than the traditional ones. Our next aim will be extending this method to several kinds of degraded samples in order to confirm the above mentioned observations.

Keywords: DNA STR typing; Forensic casework; Mentypeâ.

*Corresponding author: rismebiologia@carabinieri.it

P-252

Validation of the AmpFℓSTR® Yfiler™ kit

Romero R.E., Lizarazo R

Grupo de Genética Forense. Instituto Nacional de Medicina Legal y Ciencias Forenses.

Bogotá - Colombia

The use of Y chromosome STR markers is of grate utility in forensic cases, especially in sexual assault investigations. Recently it has been great improvement in commercial kits that offer large multiplex reactions in a single step, systems whit high discrimination power and reliable and reproducible results.

The AmpFℓSTR® Yfiler™ kit is the most recent commercial product of Appiled Biosystems that offers 17 STR from human Y chromosome DYS19, DYS385a, DYS385b, DYS389 I, DYS389 II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS448, DYS456, DYS458, DYS635, Y GATA H4, DYS438 and DYS439.

In this work several aspects were assayed. Differences in equipment used for PCR amplification, sensitivity and specificity and application on forensic cases with low concentrations of human genomic DNA.

Contact: rosa.romero@javeriana.edu.co

P-253

The genetics of pre-Roman Iberian Peninsula: a mtDNA study of ancient Iberians

Sampietro M.L¹, Caramelli  D², Lao O¹, Calafell F¹, Comas D¹., Lari M²., Agustí  B³, Bertranpetit J¹, Lalueza_Fox, C^1*

¹ Unitat de Biologia Evolutiva, Dept. Ciències de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona,Spain

² Dipartimento di Biologia Animale e Genetica, Laboratori di Antropologia, Università di Florence, Italy

³ Museu d’Arqueologia de Catalunya, Girona

^* Present address: Unitat d’Antropologia, Dept. Biologia Animal, Universitat de Barcelona, Barcelona

The Iberians developed a surprisingly sophisticated culture in the Mediterranean coast of the Iberian Peninsula from the 6th century BC to their conquest by the Romans in the 2nd century BC. They spoke and wrote a non-Indo-European language that still cannot be understood; their origins and relationships with other non-Indo-European peoples, like the Etruscans, are unclear, since their funerary practice was based on the cremation of the bodies, and therefore, anthropology has been unable to approach the study of this people. We have retrieved mitochondrial DNA (mtDNA) from a few of the scarce skeletal remains preserved, some of them belonging to ritualistically executed individuals. The most stringent authentication criteria proposed on ancient DNA, such as independent replication, aminoacid analysis, quantitation of template molecules, multiple extractions and cloning of PCR products, have been followed to obtain reliable sequences of the mtDNA hypervariable region 1 (HVR1) as well as some haplogroup diagnostic SNPs. The phylogeographic analyses show that the haplogroup composition of the ancient Iberians was very similar to that found in modern Iberian Peninsula populations, suggesting a long-term genetic continuity since pre-Roman times. Nonetheless, there is lesser genetic diversity in Iberians than among modern populations, a fact that could reflect the small population size at the origin of the population sampled and the heterogenic tribal structure of the Iberian society. Moreover, the Iberians were not specially close to the Etruscans, which points to a considerable genetic heterogeneity in Pre-Roman Western Europe.

contact: lourdes.sampietro@upf.edu

P-254

The Human Genome Diversity Project of Iran

Mohammad Hossein Sanati, M. Houshmand, M. M. Banooi, F. Mirzajani, F. Mahjoubi.

National Institute for Genetic Engineering and Biotechnology- Tehran, Iran

Iran is situated on the route to Central Asia and Turkey as well as western countries. Different ethnic groups live in Iran, among which Farsis, Kurds, Lors, Balooches, Bakhtiaris, Azari Turks, Taleshes, Turkamans, Qashqais and Arabs may be pointed out. Smaller ethnic groups also live in Iran. Turkamans, who live in Turkaman Sahara and north of Khorasan, are different from other Iranian ethnic groups in appearance, language, and culture. Qashqais, who are of Turkish origin, live in the central part of Iran. Arab clans mostly live in Khuzistan and are scattered along the coastlines of Persian Gulf.

Some groups of colored people, who are the descendants of slave trade with Zanzibar, are scattered in the southern provinces of Iran. The existing minority in the south of Iran also descends from Indian traders of past times.

Samples (1981 Individuals) from individuals within each of these populations were collected (1336 males and 612 males) and the DNA content was analyzed to produce data on the frequency of occurrence within the population of an agreed set of alleles or other genetic markers. In order to establish a resource that would be available for many years and that would allow future scientists to study any polymorphism, and in order to provide a back-up source of original sequence DNA, all blood samples were used to develop cell lines. 

Contact: massoudh@nrcgeb.ac.ir 

P-255

Peopling, demographic history and genetic structure of the Azores Islands: Integrating data from mtDNA and Y-Chromosome

Santos C^1,2, Montiel R³, Bettencourt S³, Prata MJ⁴, Abade A², Aluja MP¹, Lima M³

¹Unit of Anthropology, Department BABVE, Autonomous University of Barcelona, Spain

²Department of Anthropology, University of Coimbra, Portugal

³Center of Research in Natural Resources (CIRN), University of the Azores, Portugal

⁴Institute of Pathology and Molecular Immunology of the University of Porto (IPATIMUP), Portugal

Nine islands, clustered in 3 geographical groups, Eastern, Central and Western, form the Archipelago of the Azores. The archipelago has an area of 2 344 Km² and a total population of 237 315 inhabitants, distributed in a very asymmetric way among the three groups of islands (134 885 inhabitants in the Eastern, 98 101 in the Central, and only 4 329 inhabitants in the Western group). The Islands were discovered uninhabited by Portuguese navigators in the early 15th century. Starting from the islands of the Eastern group, the process of settlement was initiated by 1439 and lasted for over a century. Main imports came from Mainland Portugal and Madeira Island; immigrants from other European regions (Flanders, Spain, Italy, France, England, Germany, and Scotland) made up part of the first groups of settlers but, with exception of the Flemish, their overall contribution is thought to be less than that of the Portuguese groups. An African influence, derived mainly from the contribution of Sub-Saharan and Moorish slaves, is also reported. Furthermore, there is evidence that Sephardic Jews, expelled from the Iberian Peninsula, also contributed to the peopling of this Archipelago. Historical documents, however, are not sufficient to provide accurate information concerning the demographics of settlement, and do not report on how these distinct contributions were distributed among the 3 groups of islands. Genetic characterization of the Azorean population should allow the reconstruction of a more comprehensive picture of these processes. We have conducted studies to assess the variability of mtDNA and Y-chromosome markers and found that for both genetic systems the Azores Islands, as a whole, fit well into the pattern of variation described for other Western European populations. Phylogeographic analysis of mitochondrial DNA (mtDNA) showed a major contribution from Mainland Portugal as well as evidences of influxes from Northern Europeans, Africans, and Jewish groups. Characterization of Y-chromosome (NRY) markers has shown a main component of European chromosomes and also the presence of North African chromosomes in frequencies similar to those described for mainland Portugal. On the other hand, both mtDNA and Y-chromosome analysis have shown differential demographic histories for the three groups of islands forming the archipelago, especially on what concerns the Western group. This group showed a very atypical distribution of mtDNA haplogroups attributable to genetic drift but not to a differential female settlement history. However, an assessment of the NRY variability, and its comparison with mtDNA variability, evidenced a differential composition of males during the settlement of the three groups of islands, contrary to what has been previously deduced for the female settlers using mtDNA data.

Contact: mcbettencourt@notes.uac.pt 

P-256

Subtyping of D7S820 alleles in African-American population using two SNPs: rs7786079 and a new one described in this work

Sarasola E¹, González-Fernández MC¹, Ferández del Pozo V¹, Cardoso S¹, Builes JJ², Moreno MA², Bravo MLJ², Martínez de Pancorbo M¹

¹Servicio de Genómica: Banco de ADN, Universidad del País Vasco, Vitoria-Gasteiz. Spain

²GENES Ltda, Laboratorio de Genetica Forense y Huellas Digitales del DNA, Medellin, Colombia.

The locus D7S820 is located 65 bp downstream the SNP rs7786079. The design of new primers with the aim to obtain minialleles of this locus showed a new SNP in African individuals. This new SNP is located 115 bp upstream the D7S820. This work is based on the study of these two SNPs, one of them described for the first time. The aim of this study is the differentiation between D7S820 alleles carrying the same number of repetitions. The SNP rs7786079, with A and C alleles, has been studied using allele-specific polymerase chain reaction. The second one, not yet included in the databases (NCBI SNP database or www.ensebml.org) presents C and T alleles and was studied with a simple PCR followed by digestion with Sml I restriction enzyme.

Sixty four African-American samples and 34 Antioquian (Colombia) samples were analyzed. The heterozygosity of rs7786079 was 0.198 in African-American sample, whereas the Antioquia sample was A homozygous. The other SNP showed a heterozygosity of 0.167 in African-American sample and 0.019 in Antioquian individuals. These results indicate that the SNPs here studied are not discriminative of D7S820 alleles in Antioquian population. This is coincident with rs7786079 informativeness in Caucasian populations.

The disequilibrium test in African American population sample has shown that these SNP loci are closely linked, being this linkage A-C and C-T. This data is indicative that only one or both SNPs can be used to perform allelic discrimination between D7S820 alleles with the same number of repetition units.

In addition, these SNP loci do not show close linkage with D7S820 alleles, probably due to the higher mutation rate of the microsatellite loci. Taking this into account, it is possible to discriminate between D7S820 alleles combined with these SNPs. The Power of Discrimination values observed range from 0.252 to 0.315 for different alleles.

In conclusion, the genetic identification based on D7S820 locus in African American population could be improved combining the analysis of this microsatellite locus with the SNPs close to it.

contact: zobsadie@vc.ehu.es

P-257

Fetal sex determination from maternal plasma by nested PCR of the Amelogenin gene.

Sarasola E¹, Martínez de Pancorbo M¹, Martín-Vargas L², Melchor JC³, Rodríguez-Alarcón J²

¹Serviciode Genómica: Banco de ADN. Universidad del País Vasco. Vitoria-Gasteiz. Spain

²Dpto. de Pediatría, Hospital de Cruces, Barakaldo. Bizkaia. Spain

³Dpto. de Obstetricia y Ginecología, Hospital de Cruces, Barakaldo. Bizkaia. Spain

Fetal sex determination from circulating DNA in maternal plasma has been reported to be a reliable way to avoid invasive prenatal tests, such as amniocentesis or chorionic villus sampling, in pregnancies with high risk of X-linked disorders. This plasma circulating DNA is highly degraded, which makes more difficult its detection. Highly degraded DNA is common in forensic casework. The experience accumulated in manipulating forensic samples has allowed us to develop the methodology described here. The study was carried out in 22 samples from both male/female pregancies, fact which was unknown by the investigator while developing the tests. Cell-free fetal DNA was extracted from maternal plasma using phenol-cholorophorm method and the amelogenin gene was amplified by PCR. This amplification results in one fragment of 106 bp when only the X chromosome copy of the gene is present, or two fragments of 106 and 112 bp when both copies from X and Y chromosome are present. This 6 bp difference is the base of the next step, which consists in the reamplification of the previous PCR products with a primer whose 3’-end only anneals to that different 6 bp. From the 22 samples analyzed, 10 where correctly sexed as male carrying pregnancies and 11 as female carrying pregnancies. Only one female pregnancy resulted in nested PCR positive amplification. The method described here has detected one false positive, but no false negative so it could be considered a reliable approach to fetal sex determination.

contact: zobsadie@vc.ehu.es

P-258

Ancient DNA Analysis from medieval and Etruscan bones

L . Saravo*, S.Spitaleri ,  N. Staiti, E. Di Luise, C. Trapani and C. Romano

¹ Laboratory of Molecular Biology – Raggruppamento Carabinieri Investigazioni Scientifiche (RaCIS),

98128 Messina – ITALY.

DNA extraction and genotyping from ancient bones represent a great challenge for scientists investigating both on archaeological and population genetics topics. The possibility to obtain an amplifiable nuclear DNA from such items is still fully debated. Here we report our experience on some medieval and Etruscan bones

found during the excavation of different burial sites in Tuscany. Bones were extremely spoiled in outer layer but well conserved as regards to the inner tissue. Nevertheless we had to dramatically improve extraction efficiency by means of an experimental procedure. Firstly we had to purify DNA from environmental pollutants, as humic acids, and obviously from exogenous DNA. Secondly particular extraction adjustments were performed to eliminate shorter DNA fragments in order to avoid the annealing mismatch during the amplification phase. PCR was carried out by using both a 15 loci traditional STRs system and another forensic kit based on shorter STR amplicons (i.e. “Mini” STRs). Result showed that “Mini” STRs are less affected by DNA quality and/or number of copies than the traditional 15 loci STRs kit. In fact, in our experience the latter multiplex system resulted in an amplification failure whereas “Mini” STRs still provided a significant outcome. Despite the genotype profile is incomplete, it can furnish extremely important information and a promising starting point for anthropological and forensic investigation. 

Keywords: DNA STR typing; Forensic casework; Mentype, LCN..

Corresponding author: rismebiologia@carabinieri.it

P-259

Forensic ABO blood grouping by 4 SNPs analyses using ABI PRISM^® 3100 genetic analyzer

K. Satoh^1，2and Y. Itoh^1，

¹Department of Forensic Medicine, Juntendo University School of Medicine, Tokyo, Japan

² Medico-Legal Section, Criminal Investigation Laboratory, Metropolitan Police Department, Tokyo, Japan

Serologic ABO blood grouping is well established. Grouping from forensic samples such as bloodstains is usually limited to the serologic detection of ABH antigen. However, these are still some problems with establishing the grouping.  Since Yamamoto et al reported the molecular genetic basis of the three major alleles (A, B and O) at the blood group ABO gene locus, 114 alleles based on the nucleotide sequencing have been identified and described in the blood group antigen gene mutation database. We now face the problem of whether to examine all of these alleles as part of the forensic ABO blood grouping strategies.

Most of the published ABO genotyping strategies are restricted to the detection of several known ABO alleles. However, it is exceedingly important to classify four phenotypes such as A, B, O and AB to establish forensic ABO grouping by the analysis of the nucleotide sequencing. We previously proposed the analysis of four SNPs at nucleotide positions 261, 796, 802 and 803 to reflect serologic specificity.

 This paper reports effective PCR-based methods, such as sequence specific PCR with positive control (SSPPC) and confronting two pair primers (CTPP) for ABO groupings using the fragment analysis by ABI PRISM^® 3100 genetic analyzer.

 Our data showed that 105 of 114 alleles in the database corresponded to the three major alleles by assaying the 4 nucleotide positions 261, 796, 802, and 803. The remaining 9 alleles, the two Aw08 and O03 (initially called O²), and the seven O08 (initially called O³), O14 (initially called O301), O15 (initially called O302), O19 (initially called R102), O20 (initially called R103), O39 and O40 are difficult to determine whether each is involved in a A or O allele. These O alleles differ from the common O allele by the absence of one nucleotide deletion of a G at position 261. Although these frequencies are extremely low in Japan, more detailed investigation accompanied by serological prevalence data will be necessary. The common O alleles share a deletion of 261G. This deletion induces a frameshift and creates a premature stop codon. The O allele corresponds to a silent allele of the ABO gene. To determine the silent allele without deletion is difficult only by analysis of the nucleotide sequence.  Accordingly, both serologic and PCR-based testing should be applied to classify the four phenotypes, such as A, B, AB and O, in the practice of forensic ABO blood grouping.   In addition, both PCR-SSPPC and PCR-CTPP methods using fragment analysis by ABI PRISM^® 3100 genetic analyzer are an effective method, because these methods include a PCR control to examine whether the target DNA obtained from the forensic specimen can be amplified or not.

Address correspondence to:

Dr. Y.Itoh

Department of Forensic Medicine, Juntendo University School of Medicine, Hongo,

Tokyo 113-8421, Japan. TEL: +81-3-5802-1051 FAX: +81-3-5802-1050

E-MAIL: yitoh@med.Juntendo.ac.jp

P-260

PI (paternity index) vs. Residual PI in real cases. Inferences about Exclusion Power and Real Exclusion Rates over 11 STR polymorphic systems in Entre Ríos population of Argentina

Schaller LC ¹, Martínez GG ^{1 2}, Vázquez LE ¹, Bolea M ² and Martínez Jarreta B ²

¹ Servicio de Genética Forense, Superior Tribunal de Justicia, Provincia de Entre Ríos, Argentina.

² Laboratorio de Genética Forense e Identificación Humana, Universidad de Zaragoza, España.

Eleven polymorphic systems were analyzed in 107 trios compound by alleged father, mother and son. IP, Residual IP (RIP), and the respective distribution descriptive parameters were obtained. In those cases where the exclusion of paternity was determined, the exclusion percentage was evaluated for each system and it was compared with the calculated Exclusion Power. Only three IP values were observed to be inside of the curve of RIP values, although this doesn't happen for more than 99.5 % of the cases. We conclude, like other authors over other populations, that in this population more polymorphic systems must be analyzed when IP values smaller than 1000 were observed. Total Exclusion Power for this polymorphic systems was 0,99973, and D13S317 was the system with highest exclusion power (0,6183), however the highest real exclusion rate in this population was observed in F13A01 system (0,7000), calculated over the number of F13A01 exclusions above the total paternity exclusions observed.

Contact: ggmartinez@arnet.com.ar