J. Carlos Alvarez

Increasing DNA extraction yield from saliva stains with a modified Chelex method

Recovery, preservation and analysis of body fluid stains is an important aspect of forensic science. PCR-based typing of DNA extracted from recovered stains is often a crucial method to identify a perpetrator or exclude an innocent suspect. This paper reports an improved method of extracting genomic DNA from saliva stains deposited on human skin in simulated bite mark situations. Results of organic (phenol-chloroform) extraction and Chelex extraction were compared to a modified Chelex method developed by the authors. Modifications include pre-extraction preparation with proteinase K and incubations at 56 degrees C and 100 degrees C plus microconcentration of the solution. Quantification results using the classical Chelex extraction method showed that 31.9 +/- 4.22% of the deposited DNA was recovered, but using the modified Chelex extraction method DNA recovery was increased to 47.7 +/- 6.90%. The quantity and quality of extracted DNA was shown to be adequate for PCR-based typing at two STR loci.

Assessment of HV1 and HV2 mtDNA variation for forensic purposes in an Uruguayan population sample

In order to assess the utility of mtDNA typing for forensic purposes in Uruguay, a population sample of 120 maternally unrelated individuals were amplified and directly sequenced for the HV1 and for the HV2 segments in the control region of the human mtDNA, following previous international recommendations (1).

Social benefits of non-criminal genetic databases: missing persons and human remains identification

Abstract A Missing Persons Genetic Identification Program (Phoenix Program) was implemented in Spain in order to try to identify cadavers and human remains that could not be identified using traditional forensic approaches; to our knowledge, this is the first database ever implemented and in function in the world. Two separate mitochondrial DNA (mtDNA) databases have been generated and comparisons can be made automatically to match identical or similar sequences contained in both databases. One database is called the Reference Database (RD), which contains mtDNA sequences from maternal relatives of missing persons that provide the samples voluntarily after informed consent. The other database is called the Questioned Database (QD) and is comprised of mtDNA data on unknown remains and cadavers that could not be unequivocally identified. The combined database is a civil database designed solely for human identification and because of the informed consent and voluntary donation of reference samples is different from other databases now used to solve criminal cases. It is timely and incumbent on other willing countries to begin an international collaboration so compatibility and full utility can be enjoyed with this kind of non-criminal database.

Indisputable double paternity in dizygous twins

OBJECTIVE To report a case of heteropaternal superfecundation. DESIGN Case report. SETTING University paternity laboratory. PATIENT(S) Father, mother, and a set of twins. INTERVENTION(S) Blood typing conventional markers, as well as polymerase chain reaction loci and restriction fragment length polymorphism loci of DNA. MAIN OUTCOME MEASURE(S) Heteropaternal superfecundation was demonstrated after paternity investigation. RESULT(S) The probability of paternity for twin 1 was 99.9999998%, whereas that for twin 2 was excluded on the basis of the following tests: Fy, Pi, human leukocyte antigen (HLA)-DQA1, D1S80, D17S5, HBGG, D5S110, D2S44, and D10S28. CONCLUSION(S) Dizygous twins can have different biologic fathers, as demonstrated in this case. According to published data, the frequency of twins with different fathers is probably underestimated, at least in small selected populations such as those of paternity suits.

Gene-expression profiles, tumor microenvironment, and cancer stem cells in breast cancer: Latest advances towards an integrated approach

During the past decade, several high throughput analytical methods for gene-expression profiling have been developed. DNA microarrays and multiplex RT-PCR have been applied in the field of breast-cancer research to establish new molecular taxonomic classifications, or a selected group of genes able to predict the prognosis of the patients and/or their response to chemotherapy. This technology provides an opportunity to refine the anti-neoplastic treatment and avoid the currently observed under- and over-treatment of breast-cancer patients. In parallel, high throughput technologies for gene-expression analysis have been applied to research on cancer stem cells (CSCs) and the tumor microenvironment, offering a wider vision of the molecular processes that influence carcinogenic events, disease development, and the response to the treatment of breast-cancer patients. In this report, we briefly revisit the most relevant genomic studies on breast-cancer prognosis and prediction to introduce the latest advances in tumor dormancy, its implications in the clinical outcome of disease-free patients and its connection with CSCs biology and microenvironment influence in the metastatic process. Finally, we have discussed the contribution of the results of these studies to the design of new experimental strategies oriented towards personalized medicine.

Fluorescent multiplex analysis of nine STR loci: Spanish population data

A total of 171 Caucasians living in Andalucia (southern Spain) have been typed for nine short tandem repeat (STR) loci by multiplex PCR amplification using a commercially available kit (Profiler Plus; Perkin-Elmer, Norwalk, CT, USA) and semi-automatic electrophoresis (ABI Prism 377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The kit enables typing of the STR loci D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11, and D18S51. All loci, except D7S820, meet Hardy-Weinberg equilibrium. Because of the large number of loci that can be analyzed, the power of discrimination (PD) is greater than 0.99999, and the probability of exclusion (PE) reaches 0.99991 in our population sample.

Intentional Mixed Buccal Cell Reference Sample in a Paternity Case

ABSTRACT: We report a case where an alleged father (AF) attempted to substitute someone elses saliva sample for his reference sample in a paternity analysis. Buccal cells were collected from the AF and the child, and DNA analysis was performed using an autosomal STR loci (Identifiler®). The profile from the AF showed extra peaks in some loci, as well as a much higher “X” allele peak relative to the “Y” allele peak at the amelogenin locus. After conducting reanalysis by another technician with another set of positive and negative controls, it was concluded that the only source of the mixed profile was by intentional introduction by the AF, at the time of sampling, of some foreign human biological material, most likely saliva from a woman. Owing to the inconclusive results, when the AF was called back to the lab and the peculiar results were explained to him, he admitted that he had introduced into his mouth saliva from another person in an attempt to be excluded as the father of the child. Although tampering with DNA reference samples is not common, some individuals may attempt to contaminate or otherwise adulterate specimens before DNA tests. Personnel responsible for sampling should be aware of this possibility and should try to establish procedures to avoid the problem.

P.C.R. Amplification of D.N.A. from Amniotic Fluid in a Paternity Study Following a Rape

In June 1993, a 18-year old woman was raped and became pregnant. She was under psychiatric treatment because of some schizophrenic disorders associated with mental retardartion (IQ = 55). She was living with her family (parents, one brother, one sister and two maternal uncles), but nobody realized that she was pregnant until the begining of the fourth month. According to the Spanish Penal Law, this was a rape case; among other cases, having sexual intercourse with a woman who cannot give valid consent is classified as “rape”.