Simple and Sensitive Method for Identification of Human DNA by Allele-Specific Polymerase Chain Reaction of FOXP2

Abstract:  The forkhead box P2 ( FOXP2 ) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.     

Ethanol production by Candida albicans in postmortem human blood samples: effects of blood glucose level and dilution

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.     

Evaluation and usefulness of reverse dot blot DNA-PolyMarker typing in forensic case work

Experiments were performed to evaluate the Amplitype PolyMarker DNA typing system for application to forensic casework. DNA extraction using chelex was compared with phenol-chloroform extraction for various biological materials including postmortem blood, blood samples used for alcohol quantification, fresh urine, envelopes and cigarette butts. Different amounts of genomic DNA were amplified to test the sensitivity of the Amplitype PM. Mixed samples of two different bloods were typed to determine the dilution at which mixtures could be detected. Different storage conditions were evaluated using urine samples. Postmortem blood samples were typed during 4 months to determine the effects of natural degradation. A population sample of 105 unrelated individuals from South-West Switzerland was analyzed and the genotype frequencies were compared with those reported by others. Finally, practical usefulness of the Amplitype PM system is illustrated by analysing casework samples. The results of this validation proved the great usefulness and sensitivity of the Amplitype PM system using the appropriate extraction and typing method. However, mixed samples had to be interpreted with caution owing to the possibility of non-specific alleles with stored material such as urine and postmortem blood.     

PEP-PCR法及其在法医学中应用的可行性研究

提高微量检材的利用率 ,建立PEP方法并对其法医学应用进行可行性研究。用 15个随机寡核苷酸的序列作为引物 ,低特异性下扩增出微量DNA ,以此扩增物作为模扳 ,进行特异基因座扩增。经PMCT118,GABAR ,DYS19,D18S849,D3S1744,D12S10 90 ,D8S1179,D2 1S11,D18S5 1,D5S818,D13S3 17,D7S82 0 ,D3S13 5 8,vWA ,FGA ,TH0 1,CSF1PO ,TPOX ,D16S5 3 9及Amelogene等 2 0个基因座的PEP PCR与直接的PCR分析比较 ,二者分型结果一致 ,即使 1ngDNA也能得到正确分型。PEP方法可用于法医学检验 ,有利于微量物证的利用 ,使物证检材提供尽可能多的信息     

Developmental Validation of the Quantifiler® Duo DNA Quantification Kit for Simultaneous Quantification of Total Human and Human Male DNA and Detection of PCR Inhibitors in Biological Samples*

Abstract: The Quantifiler^® Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C_T) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/μL. In addition, the multiplex assay can detect as little as 25 pg/μL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFℓSTR^® Amplification Kit to obtain interpretable short tandem repeat profiles.     

Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.     

A multiplexed system for quantification of human DNA and human male DNA and detection of PCR inhibitors in biological samples

Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023-50 ng/μl. The assay was highly sensitive, detecting as little as 25 pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpF?STR^®PCR Amplification kits.     

Ethanol formation in unadulterated postmortem tissues

During the investigation of aviation accidents, postmortem samples obtained from fatal accident victims are submitted to the FAA's Civil Aerospace Medical Institute (CAMI) for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Many species of bacteria, yeast, and fungi have the ability to produce ethanol and other volatile organic compounds in postmortem specimens. The potential for postmortem ethanol formation complicates the interpretation of ethanol-positive results from accident victims. Therefore, the prevention of ethanol formation at all steps following specimen collection is a priority. Sodium fluoride is the most commonly used preservative for postmortem specimens. Several studies have been published detailing the effectiveness of sodium fluoride for the prevention of ethanol formation in blood and urine specimens; however, our laboratory receives blood or urine in approximately 70% of cases. Thus, we frequently rely on tissue specimens for ethanol analysis. The postmortem tissue specimens received by our laboratory have generally been subjected to severe trauma and may have been exposed to numerous microbial species capable of ethanol production. With this in mind, we designed an experiment utilizing unadulterated tissue specimens obtained from aviation accident victims to determine the effectiveness of sodium fluoride at various storage temperatures for the prevention of microbial ethanol formation. We found that without preservative, specimens stored at 4 degrees C for 96 h showed an increase in ethanol concentration ranging from 22 to 75 mg/hg (average 42 +/- 15 mg/hg). At 25 degrees C, these same specimens showed an increase ranging from 19 to 84 mg/hg (average 45 +/- 22 mg/hg). With the addition of 1.00% sodium fluoride, there was no significant increase in ethanol concentration at either temperature.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

Assessment of the Role Played by N‐propanol Found in Postmortem Blood in the Discrimination Between Antemortem Consumption and Postmortem Formation of Ethanol Using Rats

This study disproves the reliability of n‐propanol as a biomarker to establish whether the ethanol found in postmortem blood is derived from antemortem ingestion or postmortem putrefactive processes. Two groups of rats were given ethanol or normal saline solution, respectively, and sacrificed 1.5 h later. After putrefaction, blood and, in a few cases, urine samples from the rats were analyzed for ethanol and n‐propanol by head‐space gas chromatography equipped with flame ionization detection. Although the concentration ratios of ethanol/n‐propanol in the postmortem blood collected from the bodies without prior alcohol consumption were expected to be <20 (as per limited case reports and previous in vitro studies), in samples from several rats that were on saline solution, this ratio was found to exceed 20. In conclusion, the concentration ratio of ethanol/n‐propanol in postmortem blood does not allow for the discernment between antemortem ingestion and the postmortem synthesis of ethanol.     

Quantitative PCR overestimation of DNA in samples contaminated with tin

Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an “inhibition study” and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.     

溺死法医学检验鉴定方法评估与展望

综述近年来在硅藻检验、水中浮游生物叶绿素（A）检测、血液化学和组织化学检验等方面的最新文献报道,并对各种溺死检验方法的优缺点进行了评价：在硅藻检验中,硝酸乙醇法、破机罐法及微波消解法,可缩短检验时间,提高办案效率;酶消化法及PCR法硅藻检出率高,适用于可疑水样中硅藻密度低样品的检测。早期器官组织中浮游生物叶绿素（A）、血液和组织中其他生化指标,可作为鉴定溺死的重要参考;微量元素锶检测可用于鉴定海水中溺死;另外,硅藻及其他浮游生物遗传多态性片断PCR,可望成为新的、灵敏的溺死检测方法。     

Genotyping of DNA samples under adverse conditions of Low Copy Number—LCN (formolisados tissue samples and embedded in paraffin)

This paper aims to describe and evaluate a protocol for extraction of DNA (deoxyribonucleic acid) in formalinized tissues and embedded in paraffin for forensics genetic analysis. In outline the method is the removal of paraffin with an organic solvent in 0.3–0.5 mg of the sample of the tissue under study, followed by removal of formaldehyde, rehydration and soon after the extraction of genomic DNA. The extraction is achieved through the stages of cellular lysis, enzymatic digestion of proteins and DNA precipitation in ethanol medium. With the research we can conclude that even when the DNA is present in small quantities in conditions of extreme difficulties in its extraction, as formalinized tissues and embedded in paraffin, the technique of optimizing the extraction of DNA used both to organic extraction as Chelex, for use in the polymerase chain reaction (PCR), and possible the investigation of different samples of human tissue, biological samples, or was obtained under the conditions tested, a DNA with good quality and concentration. The samples were amplified for the mini-STRs loci using the product marketed in multilocus, using a methodology recommended by the supplier and validated for analysis of forensic DNA. Commercial kit was used MiniFiler from Applied Biosystems. The DNA fragments amplified by PCR showed that the extracted DNA had good amplification.     

The Achilles tendon as a DNA source for STR typing of highly decayed corpses

Forensic criminal casework often involves DNA profiling of human postmortem tissues, whereas degradational processes can affect PCR-based Short Tandem Repeat (STR) analysis. Degradation of DNA is observed to vary among different tissues and with time. Therefore, the stability of DNA in Achilles tendon samples is compared to that in muscle and kidney specimens with a variety of postmortem histories. Tissue samples from 28 autopsy cases, including 15 decomposed corpses and a control group of 13 nondecayed corpses were analysed. DNA was isolated using the All-tissue DNA Kit (GEN-IAL, Troisdorf, Germany), quantified by spectrophotometric measurement, amplified by the multiplex PCR genRES MPX-2 (Serac, Bad Homburg, Germany), and analysed on the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Quantitative analysis of nondecomposed tissues revealed that the recovery of DNA was highest in kidney followed by muscle, whereas Achilles tendon tissue was the poorest source of isolated DNA. Only small amounts of DNA were present in both kidney and muscle samples from decomposed corpses. However, from decayed Achilles tendon samples twice as much DNA as from nondecayed samples could be isolated on average. These results suggest DNA to be better protected in Achilles tendons. Moreover, postmortem changes in Achilles tendons may even improve DNA isolation.     

ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.     

PCR-based detection of salivary bacteria as a marker of expirated blood

Distinguishing between bloodstains caused by a spatter pattern or by expirated blood may be crucial to a forensic investigation. Expirated blood is likely to be contaminated with saliva but current techniques have limited sensitivity, especially with small bloodstains. We report that a PCR assay, designed to detect salivary bacteria, can amplify streptococcal DNA from saliva stains applied to fabrics for at least 62 days after seeding. Bacterial DNA was detected when 0.01 µl of saliva was present in the stain and the amplification was not affected by contamination with blood. These findings indicate that PCR amplification of salivary microbial DNA may have application in the identification of expirated bloodstains in forensic case-work.     

Interpreting results of ethanol analysis in postmortem specimens: a review of the literature

We searched the scientific literature for articles dealing with postmortem aspects of ethanol and problems associated with making a correct interpretation of the results. A person's blood-alcohol concentration (BAC) and state of inebriation at the time of death is not always easy to establish owing to various postmortem artifacts. The possibility of alcohol being produced in the body after death, e.g. via microbial contamination and fermentation is a recurring issue in routine casework. If ethanol remains unabsorbed in the stomach at the time of death, this raises the possibility of continued local diffusion into surrounding tissues and central blood after death. Skull trauma often renders a person unconscious for several hours before death, during which time the BAC continues to decrease owing to metabolism in the liver. Under these circumstances blood from an intracerebral or subdural clot is a useful specimen for determination of ethanol. Bodies recovered from water are particular problematic to deal with owing to possible dilution of body fluids, decomposition, and enhanced risk of microbial synthesis of ethanol. The relationship between blood and urine-ethanol concentrations has been extensively investigated in autopsy specimens and the urine/blood concentration ratio might give a clue about the stage of alcohol absorption and distribution at the time of death. Owing to extensive abdominal trauma in aviation disasters (e.g. rupture of the viscera), interpretation of BAC in autopsy specimens from the pilot and crew is highly contentious and great care is needed to reach valid conclusions. Vitreous humor is strongly recommended as a body fluid for determination of ethanol in postmortem toxicology to help establish whether the deceased had consumed ethanol before death. Less common autopsy specimens submitted for analysis include bile, bone marrow, brain, testicle, muscle tissue, liver, synovial and cerebrospinal fluids. Some investigators recommend measuring the water content of autopsy blood and if necessary correcting the concentration of ethanol to a mean value of 80% w/w, which corresponds to fresh whole blood. Alcoholics often die at home with zero or low BAC and nothing more remarkable at autopsy than a fatty liver. Increasing evidence suggests that such deaths might be caused by a pronounced ketoacidosis. Recent research has focused on developing various biochemical tests or markers of postmortem synthesis of ethanol. These include the urinary metabolites of serotonin and non-oxidative metabolites of ethanol, such as ethyl glucuronide, phosphatidylethanol and fatty acid ethyl esters. This literature review will hopefully be a good starting point for those who are contemplating a fresh investigation into some aspect of postmortem alcohol analysis and toxicology.     

微滴式数字PCR技术用于生物样品种属鉴定和绝对定量

目的 运用微滴式数字PCR技术进行生物样品的种属鉴定和绝对定量.方法 选择人mtDNA两个编码基因ND4和16S rRNA,设计特异性引物与探针,用人来源及常见动物样本验证种属特异性,再用重组质粒和2组共16份人来源生物检材,倍比稀释.使用微滴式数字PCR技术进行种属检验和绝对定量,验证其灵敏度和稳定性.结果 人重组质粒FAM （ND4）可进行人来源样品的检测,其检测结果与各级稀释梯度基本吻合,微滴式数字PCR技术可以检测出反应体系中低至单拷贝的DNA.结论 微滴式数字PCR技术可以进行生物样品的种属鉴定和绝对定量,并具有很高的灵敏度和特异性,可应用于日常法医物证检验.     

Utilizing the urinary 5-HTOL/5-HIAA ratio to determine ethanol origin in civil aviation accident victims

Specimens from fatal aviation accident victims are submitted to the FAA Civil Aerospace Medical Institute for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Care must be taken when interpreting a positive ethanol result due to the potential for postmortem ethanol formation. Several indicators of postmortem ethanol formation exist; however, none are completely reliable. The consumption of ethanol has been shown to alter the concentration of two major serotonin metabolites, 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA). While the 5-HTOL/5-HIAA ratio is normally very low, previous studies using living subjects have demonstrated that the urinary 5-HTOL/5-HIAA ratio is significantly elevated for 11-19 h after acute ethanol ingestion. Recently, our laboratory developed and validated an analytical method for the simultaneous determination of both 5-HTOL and 5-HIAA in forensic urine samples using a simple liquid/liquid extraction and LC/MS/MS and LC/MS/MS/MS. In this previous work a 15 pmol/nmol serotonin metabolite ratio cutoff was established in postmortem urine, below which it could be conclusively determined that no recent antemortem ethanol consumption had occurred. In the current study this newly validated analytical method was applied to five ethanol-positive aviation fatalities where the origin of the ethanol present could not previously be conclusively determined. In four of the five cases examined the detected ethanol was demonstrated to be present due to postmortem microbial formation, and not consumption, even though some indication of ethanol consumption may have been present.