P-201

Non-human mtDNA helps to exculpate a suspect in a homicide case

Nussbaumer C, Korschineck I

Ingenetix Ltd., Vienna, Austria

In January 2005 the dead body of a young female was found close to a highway in the South of Austria. Since the corpse had been set on fire after the woman was killed, the victim could not be identified so far. Part of the wool pullover the woman was wearing was left and showed a few short animal hairs. The investigations led the police to a young man who was already on remand due to a property offense. 

Morphological comparison of the hairs found on the pullover of the victim were suspected to derive either from an animal out of the group of minks and martens (Mustelidae) or from a dog (Canidae). Since light microscopy did not show any differences, the court gave the order to analyze the DNA of the few animal hairs collected from the victim’s pullover and to compare these hairs to the hairs found in the car the suspect had been driving and the material from the dog which belongs to the owner of the car. The question was firstly which species the hairs came from and secondly if they derive from the same individual. 

DNA from the hairs was purified using QiaAmp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol except for minor changes. The gene of cytochrome b was amplified by PCR, the products were purified and amplified by cycle sequencing using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA). Sequencing was done on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Sequence data were analyzed using Sequencing Analysis Software and SeqScape Software (Applied Biosystems).

The results revealed that all of the hairs which could be analyzed derived from the species Canis familiaris (dog). Only one single hair, found in the trunk of the car, came from Felis catus (cat). Since only very few of the hairs showed hair roots, STR analysis for identification of the individual could not be applied. Thus, the second question had to be answered by further analysis of the mtDNA. Although the hypervariable region (HV) in the non-coding control region of the mtDNA in dogs is not as variable as in human it is used to compare individuals within this species. A 350 bp product within the region HV1 was amplified from all samples. All the hairs from the pullover of the victim showed the same sequence. The hairs from the car showed two different mtDNA haplotypes. These sequences differed (at least 2 mutations) from the sequence of the hairs found on the victim’s pullover. The sequence of the dog of the owner of the car matched the sequence of one of the two haplotypes found in the car. The other haplotype derived from an unknown dog.

Thus, we were able to show, that the hair from the victim’s pullover doesn’t belong to the dog associated with the suspect. This evidence amongst other investigations of the police helped to exculpate the young man. Neither the identity of the victim nor the identity of the murderer could be resolved up to the present.

Contact: christa.nussbaumer@ingenetix.com

P-202

Simultaneous detection of DNA length and sequence variations by liquid chromatography electrospray ionization time-of-flight mass spectrometry

Oberacher H¹, Niederstätter H¹, Casetta B², Parson W¹

¹Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria

²Applied Biosystems, Monza, Italy

The combination of ion-pair reversed phase high-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry is presented as an efficient method for the fast and accurate detection of sequence variations. The chromatographic separation enables the highly efficient purification of nucleic acids prior to their mass spectrometric analysis. Therefore, sample preparation is limited to PCR only. As separations are performed at elevated temperatures (65-80°C), liquid chromatography represents an elegant way for denaturing double-stranded nucleic acids into the corresponding single strands. The denaturation is advantageous because of the division in half of the masses of the detected species and the possibility to identify base substitutions by measuring mass differences ranging in size between 9.01 and 40.02 amu, which would have been missed by measuring the nearly composition independent molecular masses of double-stranded nucleic acids. Furthermore, as the allelic state derived from one single strand is confirmed by the result obtained from the complementary single strand, the reliability of the mass spectrometric genotyping assay is increased. Taking advantage of the high mass spectrometric performance of the time-of-flight mass analyzer all kind of single base exchanges were detectable in PCR amplicons with lengths up to approximately 250 base pairs. Consequently, the described hyphenated technique represents one of the most powerful mass spectrometric genotyping assays available today.

The mass spectrometric genotyping assay was applied to the characterization of two simultaneously amplified PCR products covering the homopolymeric stretches of cytosines within the hypervariable regions 1 and 2 of the non-coding mitochondrial control region. Based on the high performance of the assay length and sequence variants were simultaneously identified. Additionally, relative quantification of the individual allele frequencies was obtainable by measuring and comparing individual peak intensities. Mass spectrometric results were checked by sequencing.

Contact: herbert.oberacher@uibk.ac.at 

P-203

Population Study of Four X Chromosomal STR Loci in the UK Population

Oguzturun C, Thacker CR, Ballard D, Syndercombe Court D

Centre for Haematology, ICMS, Barts and The London, Queen Mary's School of Medicine and Dentistry, UK

X-chromosome STR typing can complement existing DNA profiling being carried out by laboratories and can also offer very useful information in cases of complex kinship analysis.  In this study a database relevant to the UK population was compiled.  The four X-chromosome short tandem repeats DX8378, DX7132, DX7423 and HPRTB were used to generate allele frequencies in a population sample of 600 unrelated males and females from the UK population.  The population was composed of three subsets of data: 200 individuals who described themselves as Irish Caucasian (originating from and resident in Southern Ireland); 200 individuals who described themselves as British Caucasian (originating from and resident in mainland Britain) and 200 individuals who described themselves as South Asian (originating mainly from the countries of Bangladesh and Pakistan, resident in mainland Britain).  Amplification was performed using the Mentype^® Argus X-UL PCR amplification kit (Biotype AG) which enabled the four X chromosome markers and the sex marker Amelogenin to be amplified simultaneously.  Slight modifications were made to the amplification conditions recommended by the manufacturer.  Products were detected using ABI PRISM^® 3100 Genetic Analyzer (Applied Biosystems).  Allele frequencies were calculated and the three population subsets compared. The Mentype^® Argus X-UL PCR amplification kit was found to be a robust system and a useful addition to autosomal markers routinely used in forensic and paternity testing applications. 

Address for Correspondence:

Catherine R Thacker

Centre for Haematology

Institute of Cell and Molecular Science

Barts and The London

Queen Mary’s School of Medicine and Dentistry

4 Newark Street

London E1 2AT

United Kingdom

E-mail: c.r.thacker@qmul.ac.uk

P-204

Preliminary studies of individual genetic identification of domestic dogs

(Canis familiaris)

Oliveira AC¹, Balsa F¹, Brito P¹, Lopes V¹, Serra A¹, Carvalho M¹, Anjos MJ¹, Andrade L¹,

Corte-Real F², Vide MC¹

¹Forensic Genetic Service. National Institute of Legal Medicine. Largo da Sé Nova, 3000 Coimbra. Portugal

²National Institute of Legal Medicine. Largo da Sé Nova, 3000 Coimbra. Portugal

Human traces are not the only source of biological samples that can be collected from a crime scene. Biological traces from cats, dogs and other domestic animals can be found related to suspects or victims being themselves the protagonists of the forensic case.

According to Portuguese Legislation (D.L. 313/2003 of 17 of December art 6^th) is obligator the individual identification of dogs dangerous or potentially dangerous.

It seems important to follow this subject with the implementation of genetic identification.

So, this work has the aim to study STR's of DNA for individual genetic identification of dogs.

DNA was extracted by Chelex™100 (Walsh, et al) from saliva of different dogs races. The DNA was amplified 10 STR loci with StockMarks^® Kit - Canine Genotyping - PEZ 1, FHC 2054, FHC 2010, PEZ 5, PEZ 20, PEZ 12, PEZ 3, PEZ 6, PEZ 8 and FHC 2079.

The detection was carried out on ABI Prism™ 310 Genetic Analyser with internal standard (Rox 350), the DNA sample control and an allelic cocktail.

Contact: geneforense@dcinml.mj.pt 

P-205

Outcome in acute lymphobastic leukaemia: influence of thiopurine methyltransferase genetic polymorphism

Oliveira E^1,2; Alves S³; Quental S¹, Ferreira F⁴; Norton L⁵; Costa V⁵; Amorim A^1,2; Prata MJ^1,2;

¹IPATIMUP, Porto, Portugal

²Faculdade de Ciências da Universidade do Porto, Porto, Portugal

³Unidade de Enzimologia, Instituto de Genética Médica Jacinto Magalhães, Porto, Portugal

⁴Serviço de Hematologia Clínica, Hospital Geral S. João, Porto, Portugal

⁵Serviço de Pediatria, Instituto Português de Oncologia, Porto, Portugal

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer accounting for 25-30% of all childhood malignancies. Presently, due to the introduction of multiagent chemotherapy, the cure rate nearly reaches 80%. However, a wide inter-patient variability in tolerability to drug regimens does exist and these differences highly influence the success of ALL therapy. One current treatment strategy for improving cure rate is to modify the intensity of drug dosages, but usually the genetic host characteristics are not used for assigning ALL patients to risks groups or for individually tailoring the therapy.

One of the best known functional polymorphisms in genes involved in ALL drugs action or metabolism is that in thiopurine methyltransferase (TPMT), for which the impact of TPMT genotypes in 6-mercaptopurine (6MP) toxicity is well documented.

In order to assess the influence of this polymorphism in ALL outcome in patient from North Portugal, we have used PCR/HCSGE based methods to characterise molecularly a sample of 110 children with ALL who were diagnosed and followed-up in Hospital de São João or IPO, both from Porto. Four distinct alleles associated with TPMT deficiency, TPMT*3A, *3C, *2 and *8, were found in heterozygous individuals representing 11.8% of the sample.

Several parameters related with ALL outcome were compared in subsamples of children homozygous or heterozygous for TPMT. In the heterozygous group, 6MP dosages were lower comparatively to the other group, and higher the number of whole interruptions during treatment or interruptions due to haematological toxicity as well as the number of relapses or deaths.

These findings qualitatively replicate previous reports relating TPMT with a poor outcome in ALL, although probably due to the low sample size of our study, differences between the two groups do not reach statistical significance.

Since TPMT represents a determinant of 6MP response and ALL outcome, this study reinforce the relevance of introduce the prospective analysis of TPMT prior to any ALL treatment, in order to individually optimise 6MP therapy and avoid adverse reactions to this drug.

Contact: mquental@ipatimup.pt 

P-206

Power of Exclusion of 18 autosomic STR loci in a Brazilian Center-West region population sample

Oliveira SF¹, Trindade-Filho A², Mendes CRBO², Paula KAA², Maia FAS², Pak HI², Dalton GC²

¹Departamento de Genética e Morfologia, Universidade de Brasília, Distrito Federal, Brazil

² Forensic DNA Research Institute, Policia Civil do Distrito Federal, Distrito Federal, Brazil.

The purpose of this work was analyze the Power of Exclusion (PE) in paternity investigation cases from Distrito Federal of Brazil (Center-West region of Brazil) using a local population sample in comparison with Promega Corporation published databank. We used 300 cases where the alleged father was excluded and a databank of 917 individuals from Distrito Federal, both analyzed for eighteen STRs loci (D16S539, D7S820, D13S317, D5S818, CSF1PO, TPOX, TH01, vWA, Penta-E, D18S51, D21S11, D3S1358, FGA, D8S1179, F13A01, FESFPS, F13B e LPL). The expected and observed values in the paternity exclusion cases were compared for each locus and then for each multiplex system employed (PowerPlex 1.1, PowerPlex 2.1 and FFFL). The number of times that each locus was used in the exclusion paternity cases implicated in the number of times it participated in the exclusion (observed value) which was compared with the expected value, according to its Power of Exclusion using both databanks: the population sample from and the one by Promega Corporation. The values of c² show a P higher than 0.05 for each locus, except in TPOX and D16S539 (0.05 > P > 0.01). For both loci in both databanks, the observed number of exclusions was considerably higher than the expected ones. Complementarily, the combined Power of Exclusion obtained for each multiplex system for each databank was compared and results showed no significant difference.

Contact: silviene@unb.br

P-207

Characterization of a novel variable number of tandem repeats (VNTR) polymorphism in CIAS1 gene

Omi T^1,2, Kumada M^1,2, Okuda H^1,2, Gotoh T^1,2, Kamesaki T^1,2, Kajii E^1,2, Sakamoto A¹ and Iwamoto S^1,2

1. Division of Human Genetics, Center for Community Medicine, Tochigi, Jichi Medical School, Japan

2. Division of Legal Medicine, Center for Community Medicine, Tochigi, Jichi Medical School, Japan

Cold-induced autoinflammatory syndrome 1 (MIN 606416; CIAS1) gene encodes cryopyrin/NALP3 (NACHT-LRR-PYD-containing protein-3)/PYPAF1 (PYRIN-containing Apaf1-like protein) protein, predominantly expressed in peripheral blood leukocytes. The function of these proteins is to regulate apoptosis or inflammation through the activation of NF-B and caspase. Recent genetic analyses showed an association between inflammation and oxidative stress-related genes in the development of hypertension. We performed the single-candidate-gene approach study of CIAS1 for essential hypertension and identified a novel VNTR polymorphism in this gene. The VNTR polymorphism was consisted of a 42-bp repeat core sequences in the CIAS1 intron 4 (CIAS1 42bp–VNTR). Four novel alleles containing 12, 9, 7, and 6 repeats were detected with frequencies of 0.577, 0.008, 0.248, and 0.167 from the 507 unrelated Japanese individuals, respectively. Case-control study showed that the frequency of 12-12 genotype was significantly higher in 1087 patients with hypertension compared with 1033 control subjects (P=0.007; Odds ratio=1.24). Association study between the VNTR genotype and blood pressure revealed that the systolic blood pressure level of 12-12 subjects was significantly higher in the random population (n=285, men, P=0.009).  The real time PCR analysis showed that among healthy young adults, 12-12 subjects expressed CIAS1 mRNA in peripheral leukocytes significantly more abundantly than X-X (X: 9, 6, and 7) subjects (P<.05). Reporter gene assay of the CIAS1 42bp-VNTR in HL60 stimulated by lipopolysaccharides showed that the intronic sequence involving 12 repeat increased the expression of luciferase compared with 9, 7, and 6 repeats. These results suggest that the CIAS1 42bp-VNTR modifies the expression level of the CIAS1 transcript. Thus, we propose that CIAS1 is a new candidate for the hypertension-susceptibility locus.

Contact: t-omi@jichi.ac.jp

P-208

Y-chromosome genetic structure in a sub-Apennine population of  the Marches (central Italy): analysis by SNP and STR polymorphisms.

Onofri V, Alessandrini F, Buscemi L, Pesaresi M, Turchi C, Tagliabracci A

Institute of Legal Medicine, Università Politecnica delle Marche, Ancona, Italy

The male-specific region of the Y chromosome (MSY) region spans many repetitive (STR) and sequencing (SNP) Y-markers, which represent a precious tool for both human evolutionary studies and forensic identification purposes.

This study was carried out on subjects living in Fabriano and Urbino, two small towns in the upland area of the Marches, speaking different dialects and submitted to a limited genetic flow. The origins of these two micro-populations might be the long-term divergence from a common group of settlers in the Apennine mountains of the Marches region, or separate evolution from other geographical areas of the Italian peninsula.

The aim of this work was to compare the genetic structure of Y-chromosome in the Apennine populations with other Italian populations in order to obtain information on the settlement of  the Marches.

81 healthy male donors, unrelated and with different surnames, were selected from two hinterland areas of the Marches region, 44 from Fabriano and 37 from Urbino. DNA was extracted from whole peripheral blood using phenol-chloroform protocol. 37 Y-SNPs were analyzed using primer extension reaction. Two multiplexes - arbitrarily named MY1 and MY2 - were developed to explore the basal branches of the tree encompassing all the major clades A-R: MY1 for markers M35, M89, M172, M170, M9, M173, M45; and MY2 for markers M52, M216, M174, M181, M201, M91, M96, M214. To obtain a more discriminative haplogroup assignment, a large number of the shallowest SNPs for the most frequent haplogroups in Italy were selected: R1, E3b, J2 and I. Four distinct multiplex PCRs were set up, capable of typing the more superficial branches typical of these haplogroups, named MY-E3b (M78, M107, M224, M165, M148, M81), MY-J2 (M158, M68, M47, M102, M137, M67), MY-R1 (M17, M269, M18, P25, SRY10831.2) and MY-I (M72, M223, M26, M21, M161).

All samples were genotyped for Y chromosome microsatellites using AmpFlSTR® Yfiler^TM (AB) that allow the co-amplification of the core set of European Minimal Haplotype, and other eight loci (DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and Y GATA H4).

Pairwise differences among haplogroups, gene frequency and haplotype diversity were estimated.

contact: a.tagliabracci@univpm.it

P-209

A comparative study of STRs and SNPs typing efficiency in highly degraded forensic samples

Onori N, Onofri V, Alessandrini F, Buscemi L, Pesaresi M, Turchi C, Tagliabracci A

Institute of Legal Medicine, Università Politecnica delle Marche, Ancona, Italy

DNA recovered at a crime scene often results as damaged; this represents enormous difficulty for the correct typing because of fragmentation or the lack of DNA region of interest. It is well known that DNA is subject to enzymatic and chemical post-mortem damage: DNasi I randomly cuts the histon-free double strand DNA and hydrolysis, oxidation, alkylation and cross-linking phenomena produce alterations at single nucleotides, hindering DNA polymerase activity or introducing mismatches.

The STR markers commonly used in forensic casework give only partial positive results with degraded samples, yielding a discrimination power not sufficient for a certain identification. Forensic geneticists have recently focused their attention on SNPs, which can be analysed in short amplicons, thus allowing typing of degraded DNA.

No experimental studies exist that have monitored STRs and SNPs genotyping efficiency during the DNA degradation process. For this reason, a set of biological samples was prepared in order to simulate the effects of natural DNA degradation on: dry and wet bloodstains, 20g of male muscle tissue stored in the open air, buried, immersed in river and sea water. The DNA was extracted by the phenol-chloroform method every month for nine months and electrophoresed on agarose gel in order to evaluate the fragmentation degree. Human DNA quantification was accomplished by Quantiblot (AB). All DNA samples were submitted to amplification of nuclear microsatellites, using AmpFlSTR Identifiler PCR Amplification kit (AB), and Y chromosome biallelic polymorphisms, employing a set of multiplex PCRs developed in our laboratory.

As expected, microsatellite analysis showed a progressive allelic and locus drop-out for the higher molecular weight STR loci in all DNA samples. SNPs amplification reactions gave results depending on the different sample type and storage conditions, and a nucleotide alteration was also observed for the locus M269.

The results suggest that further studies are necessary to establish the biochemical alterations which can affect SNP allele typing, the frequency of their occurrence and the consequent implications in forensic science.

contact: a.tagliabracci@univpm.it

P-210

The Y-chromosome in the Azores Islands: phylogeny and diversity

Pacheco PR^1,2, Branco CC^1,2, Cabral R^1,2, de Fez L^1,2, Araújo AL³, Peixoto BR^1,2, Mendonça P³, Mota-Vieira L^1,2

¹ Molecular Genetics and Pathology Unit, Hospital of Divino Espirito Santo, São Miguel, Azores, Portugal

² Instituto Gulbenkian de Ciência, Oeiras, Portugal

³ Hematology Department, Hospital of Divino Espirito Santo, São Miguel, Azores, Portugal

The Azores, a Portuguese archipelago located in the North Atlantic Ocean, had no native population when the Portuguese first arrived in the 15th century. The islands were populated mainly by the Portuguese, but Jews, Moorish prisoners, African slaves, Flemish, French and Spaniards also contributed to the initial settlement.

To understand the paternal origins and diversity of the extant Azorean population, we typed genomic DNA samples from 172 individuals using a combination of 10 Y-biallelic markers (YAP, SRY-1532, SRY-2627, 92R7, M9, sY81, Tat, SRY-8299, 12f2 and LLY22g) and the following Y-chromosomal STR systems: DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393 and DYS385 a/b.

We identified nine different haplogroups, most of which are frequent in Europe. Haplogroup J* is the second most frequent in the Azores (13.4%), but it is modestly represented in mainland Portugal (6.8%). The other non-European haplogroups, N3 and E3a, which are prevalent in Asia and sub-Saharan Africa, respectively, have been found in the Azores (0.6% and 1.2%, respectively) but not in mainland Portugal. A Y-chromosomal haplotype was constructed for each individual using the seven loci. In total, 118 different haplotypes were observed in the 172 sample set (68.6% discriminatory capacity). Haplotype diversity value was high (0.9994), due to high variability of the Y-STRs. Moreover it is important to notice the great genetic diversity observed within the Azorean group. Microsatellite data indicate that the mean gene diversity (D) value for all the loci analysed in our sample set is 0.590, (values range from 0.4592 for DYS393 to 0.8212 for DYS385).

Taken together, our analysis suggests that the current paternal pool of the Azorean population is, to a great extent (59,3%), of Portuguese descent with significant contributions from people with other genetic backgrounds

contact:  paularpacheco@hdes.pt

Funded by DRCT, Azores

P-211

Identification of the Finnish Tsunami Victims

Palo JU, Hedman M, Sajantila A

Department of Forensic Medicine, University of Helsinki, Helsinki, Finland

The Asian Tsunami on December 26^th 2004, has been estimated to have caused the death of more than 173 000 people in eleven countries around the Indian Ocean. Among the casualties were 178 Finnish tourists, belonging to 75 extended families. As numerous complete families were affected, the age distribution of the missing persons is clearly bimodal with peaks around ten and forty years. The identification process of the Finnish victims, led by the Disaster Victim Identification (DVI) team of the National Bureau of Investigation, started on Dec 28^th. Large amount of diverse AM data has been collected by the local section of the team, including DNA reference samples from surviving relatives and personal items. Four months after the catastrophe, 117 of the victims have been identified and repatriated solely with the aid of AM dental records and fingerprints. However, majority of the missing children lack conclusive dental AM data due to intact teeth. This has led to a bias in the age distribution of the identified victims – 75% of the persons missing after four months of the disaster were under 18 years of age. For these, the identification can be achieved only through DNA-based methods. As direct DNA profiles are largely lacking, reference samples from the relatives were required. Graphical pedigrees constructed at the early phase of the identification process have helped in planning effective sampling. The closest relatives for most of the missing children were among the victims, therefore PM femur bone samples have been taken in the autopsies performed for all repatriated bodies, immediately followed by DNA extraction and profiling for sixteen STR loci. The careful sample handling and phenol-chloroform-based DNA extraction has ensured a very high success rate in recovering the DNA profiles. The data handling, current state of the identification as well as the relative importance of different types of data for the identification will be discussed.

Contact: jukka.palo@helsinki.fi 

P-212

Improved Y-STR analysis of degraded DNA using reduced size STR amplicons

Myung Jin Park¹, Ji-Eun Yoo¹, Ukhee Chung¹, Hwan Young Lee¹, Chang-Lyuk Yoon^2,3, Kyoung-Jin Shin^1,3

¹Department of Forensic Medicine, College of Medicine, Yonsei University, Seoul, Korea

²Department of Oral Medicine and Forensic Odontology, College of Dentistry, Chosun University, Kwangju, Korea

³ Human Identification Research Institute, Yonsei University, Seoul, Korea

To increase PCR sensitivity in amplification of degraded DNA, we have developed two new multiplex PCR sets for 17 Y-STR loci (DYS19, DYS385a/b, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, GATA C4 and GATA H4) by reducing the sizes of some amplicons in a commercial Y-STR kit, AmpFlSTR^® Yfiler™ (Applied Biosystems). Among 17 Y-STR loci, DYS385a/b, DYS390, DYS391, DYS392, DYS438, DYS439, DYS448 and GATA C4 have been redesigned to produce reduced size amplicons by moving primers as close as possible to the STR repeat region. DYS393, DYS456, DYS458 and GATA H4 have small PCR product sizes (99~165 bp) in AmpFlSTR^® Yfiler™, and newly designed PCR primers for those STRs in the new multiplex also generated amplicons of similar sizes. In addition, we redesigned the amplification primers for DYS19, DYS389-I, DYS389-II, and DYS437 which have relatively large PCR products (150~286 bp) in AmpFlSTR^® Yfiler™, but they could not be made to generate amplicons of smaller sizes than those of AmpFlSTR^® Yfiler™. We performed concordence study for 100 unrelated Korean samples, and the genotypes obtained from two new multiplex were the same as the genotypes obtained from AmpFlSTR^® Yfiler™ kit. To assess the effectiveness of new multiplex, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to AmpFlSTR^® Yfiler™. We also conducted sensitivity test in blood genomic DNA, and this showed that all Y-STR loci in two new multiplex were reliable and sensitive at template concentrations as low as 31 pg/10 μl. Moreover, comparison studies in 30 samples of 50-year old skeletal remains demonstrated that the multiplex were capable of producing more complete profiles in comparison with AmpFlSTR^® Yfiler™. Overall, our data verified that two new multiplex can provide fully concordance results to commercial STR kits and can produce improved signal from degraded DNA than AmpFlSTR^® Yfiler™.

Contact: hylee192@yumc.yonsei.ac.kr

P-213

Organizing The Argentinian Combined DNA Index System (CODIS)

Penacino GA

Sociedad Latinoamericana de Genetica Forense (Latin American Society for Forensic Genetics): www.slagf.org

Unidad de Analisis de ADN (DNA Analysis Unit) - www.adn.ac - COFyBCF - Rocamora 4045, Buenos Aires, ARGENTINA - Phone/fax (5411) 4862-1142 - E-mail presidente@slagf.org

As proposed our Latin American Society for Forensic Genetics in June 17th, 2003, last year the Argentinian Minister of Justice, Security and Human Rights signed the Resolution # 415/2004 (see the full text in spanish in www.slagf.org), that create the DNA Fingerprint Registry, similar to American CODIS.

The Registry blends computer and DNA technologies into an effective tool for fighting violent crime. The system uses two indexes to generate investigative leads in crimes where biological evidence is recovered from the crime scene. The Convicted Offender index contains DNA profiles of individuals convicted of felony sex offenses (and other violent crimes). The Forensic index contains DNA profiles developed from crime scene evidence. The Registry utilizes computer software to automatically search these indexes for matching DNA profiles.

Like the American CODIS, this is a system of pointers; the database only contains information necessary for making matches. Profiles stored contain a specimen identifier, the sponsoring laboratory's identifier, the initials (or name) of DNA personnel associated with the analysis, and the actual DNA characteristics. Matches made among profiles in the Forensic Index can link crime scenes together; possibly identifying serial offenders. Based on a match, police can coordinate separate investigations, and share leads developed independently. Matches made between the Forensic and Convicted Offender indexes ultimately provide investigators with the identity of the suspect(s).

Following are the major enhancements planned for next years:

·        There are differences among province laws, so the first step is to make compatible them.

·        Optimize software performance for Local, Province and National Indexes.

·        Select the labs with high-quality standards. Quality controls in Latin America are conducted by the Latin American Society for Forensic Genetics and the Spanish and Portuguese Speaking Group of the ISFG.

·        Training courses to DNA analysts from participating laboratories.

·        Begin operation of the National DNA Index System.

·        Proliferate the installed base to include all crime laboratories performing DNA analysis.

Contact: gpenacino@wwiecorp.com 

P-214

Development of a bidirectional exchange between the Sapphire LIMS and analytical softwares to drastically increase the throughput of a forensic laboratory

Pene L, Barsacq S, Gleizes A, Paléologue A

Laboratoire Police Scientifique de Lyon

During the recent years, in France, different laws extended the number of offences categories that are concerned by the DNA national database. This extension of the DNA database has led to an increase of the number of caseworks and database samples that the french laboratories have to analyse. In order to ensure the analysis of this increasing samples number, we received, in 2004, a grant, dedicated to buy new analytical devices and a Laboratory Integrated Management System (LIMS). The LIMS, implemented in the laboratory, is a Labvantage product called Sapphire, distributed in France by SpectraLIMS. Based on the practical experience of a first home-made LIMS and the current use of robotic platforms since four years, we have decided to design a global project that both automate the processing of analytical data and samples. We would like to reach a medium throughput: 15000 database samples and 21000 casework samples analysed per year. In order to achieve this goal, we are trying to automate all the repetitive tasks for the laboratory members where the error risk is high. Moreover, we are trying to use all the flexibility and subtlety wich offer the use of a LIMS that exchanges data with analytical softwares. We are going to illustrate our philosophy with examples along the description of the database samples analytical pathway. In our analytical pathway, all the reference samples must be on FTA paper to be analysed. Two punches of one FTA card are distributed in two wells of two different FTA plates, thanks to a puncher. The output file of the puncher is imported in the LIMS which triggers the creation of an "FTA plate". When the object "FTA plate" is created in the LIMS, different analysis are associated with this object. One analysis defines one step which undergoes the "FTA plate". All the steps are automatised on pre-PCR and post-PCR platforms, thanks to input-files, called work-lists. These work-lists define the treatment of each well of the plates. In order to check the quality of robotic platform work, out-put files are created at the end of robotic method. These files are attached in the LIMS to the object "FTA plate". In order to analyse the FTA amplicons, a second plate object is created in the LIMS : the "Electrophoresis Capillary plate" ("EC plate"). The analyst has the choice to merge different complete "FTA plates" in one "384 EC plate" or to pick-up few samples of "FTA plates"to dispense in one "96 EC plate". This last option allows to re-migrate, on the genetic analyser, only the samples wich fail during the first analysis. This possibility is very convenient to optimize the use of multicapillary genetic analyser. Following the electrophoresis, a GeneMapper project is generated with sample files grouped by "FTA plate" origin or "EC plate" origin. The GeneMapper software affects numerous quality factors for each locus genotyped. The importation of alleles in the LIMS is conditioned by the quality factors associated with each locus. Moreover, the LIMS affects a label for each global genotype. All this importation algorithm allows the analyst to only review, in a final table, the genotype loci that are not automatically labelled by the GeneMapper analysis method.

Contact: laurent.pene@laposte.net