Miguel Lorente

An Improved Method to Recover Saliva from Human Skin: The Double Swab Technique

Human bite mark evidence is often found in violent crimes. Due to the difficulties of physically comparing an injury site on elastic and curved skin surfaces to the teeth of a suspect, the authors have considered using salivary DNA evidence to identify the bite perpetrator. Several techniques were evaluated to determine the best method of recovering saliva from human skin before extracting genomic DNA from the collection substrate. A classical stain recovery technique using a wet cotton swab was tested against one utilizing a wet filter paper. Additionally, a new method, referred to as the double swab technique, using a wet cotton swab followed by a dry cotton swab was also evaluated. After recovering a dried saliva stain, DNA was extracted using the modified Chelex method, quantified using the slot-blot procedure, and amplified at three polymorphic loci. The double swab technique showed the highest percentage recovery of saliva from human skin among the three methods studied. This technique is suggested as an improvement over the classical single wet cotton swab technique.

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.

PCR-Based DNA Typing of Saliva Stains Recovered from Human Skin

Human bites in cases of homicide, sexual assault, and abuse are often distorted due to the elasticity and curvature of the skin. Physical comparison of a bite mark to a suspects teeth is sometimes difficult. Saliva, which is usually deposited during biting, can be collected and analyzed to identify the perpetrator. Using simulated bite mark situations in two experimental series, three samples of 40 microL of whole saliva were deposited on the skin of 27 cadavers (at 33 sites) and three samples of 100 microL of whole saliva were deposited on the skin of 5 cadavers (at 12 sites). Saliva was collected using the double swab technique at t = 5 min, t = 24 h, and t = 48 h. DNA was extracted using the modified Chelex method and submitted to PCR-based typing at two short tandem repeat loci. Results indicate that the concentration of DNA in saliva recovered from skin varies as a function of time since deposition. There is a significant decrease in concentration in the first 24 h but the concentration remains stable from 24 to 48 h. The success of PCR amplification is independent of the time since deposition or the concentration of DNA in the saliva sample. Contamination from the DNA of the cadaver was not found in any of the cases studied.

Analysis of short tandem repeat (STR) HUMVWA in the Spanish population

Amplification by polymerase chain reaction (PCR) of variable number of tandem repeat (VNTR) loci and subsequent typing by electrophoresis and silver staining has become a useful tool for identity testing. One viable group of genetic markers amenable to amplification by PCR is the short tandem repeat (STR) loci. A horizontal discontinuous polyacrylamide gel electrophoresis (PAGE) method was used to type the amplified products of the STR HUMVWA. Typing for VWA of 120 unrelated Spanish Caucasians was done. Six alleles were observed with frequencies in the range 0.096-0.242. The genotype distribution meets Hardy-Weinberg expectations (0.25 < P < 0.50). The heterozygosity was 73.3% and the discrimination power (DP) 0.94. Simultaneously, in a small sample of families (n = 24) no new mutations could be found.

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.

Dandruff as a Potential Source of DNA in Forensic Casework

Dandruff is a clinical alteration of the skin that consists histologically of orthokeratotic clumps with minute parakeratotic foci found in inflammatory pathologies such as seborrheic dermatitis and psoriasis. Therefore, some nucleated cells should be found in dandruff and hence there is a possibility that forensically typeable DNA could be extracted from dandruff. Because of a particular case in which we were involved, a study was carried out to determine whether or not DNA could be extracted from dandruff, and if the two most widely used extraction techniques (Chelex and organic) would be applicable. Results show that sufficient quantities of DNA (more than 30 to 40 ng) can be obtained from as little as 1.0 to 1.5 mg of dandruff. Both methods yield DNA, although the organic procedure seems to yield more (72.5 ng Chelex vs. 183.3 ng organic). All the DNA samples extracted were typed correctly for the loci HUMTH01 and HUMvWA. Therefore, dandruff can be considered a potential source of DNA for forensic identification.

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.

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.

Spanish population data on seven loci : D1S80, D17S5, HUMTH01, HUMVWA, ACTBP2, D21S11 and HLA-DQA1

Blood samples from 120 Spanish Caucasian individuals were amplified and typed by electrophoresis at six loci, and by reverse dot-blot hybridization at one locus. Results demonstrate the assumption of independence within and between the seven loci analyzed. Therefore, a Spanish population database has been established and statistical analysis shows that a high degree of discrimination can be obtained when all seven (or fewer) loci are used to characterize forensic biological evidence.

Analysis of the HUMTH01 Allele Frequencies in the Spanish Population

Genetic marker typing based on DNA amplification by the polymerase chain reaction (PCR) increasingly is being employed in forensic casework and for paternity testing. The allele frequencies were determined using PCR for 120 unrelated Spanish Caucasians for the locus HUMTHOH1. Six alleles were observed, with frequencies ranging from 0.013 (allele 11) to 0.254 (allele 10). The observed heterozygosity was 75.8%, and the power of discrimination is 0.92. The genotype distribution meets Hardy-Weinberg expectations.