Validation of a DNA Quantitation Method on the Biomek® 3000

Abstract: Laboratory automation has the ability to increase the throughput and efficiency of laboratory processes to keep pace with current backlogs and requests for analysis. This paper addresses the specific studies employed to properly evaluate an automated method for DNA quantitation setup using Applied Biosystems Quantifiler? Human DNA Quantification kit on a Biomek^® 3000. The calibration of robotic pipetting as well as comparison with manually performed steps confirmed the accuracy of the automated methods used. Reproducibility studies evaluated differences between robotic and manually prepared human DNA standard curves. Additional studies examined DNA samples of known quantities, extract storage formats, sensitivity, and an assessment of contamination. The Biomek^® 3000 not only demonstrated reproducibility and accuracy that equaled or surpassed the manual method but also revealed a contamination‐free method to replace the multiple pipetting steps required during quantitation setup.     

A New Method for Human ABO Genotyping Using a Universal Reporter Primer System

Abstract: We developed a new method for forensic ABO genotyping based on a universal reporter primer (URP) system. This allows for the simultaneous detection of six single nucleotide polymorphism (SNP) sites in the ABO gene (nucleotide positions 261, 297, 526, 703, 796, and 803). This URP system provides obvious peaks, ranging from 82 to 151 bp in length. ABO genotypes were classified and successfully genotyped by our method, including minor alleles that may cause a discrepancy between the genetic data and serological phenotypes. Full profiles were identified using as little as 0.1 ng (0.05 ng/reaction) of standard K562 and 9947A DNA. Moreover, the success rate of genotyping from a URP system was much higher than that from a conventional primer extension method in degraded DNA. This method enables simple and rapid detection of multiple SNP sites on human ABO genes and is highly specific and sensitive when using limited and degraded DNA.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

Investigation of Reproducibility and Error Associated with qPCR Methods using Quantifiler® Duo DNA Quantification Kit*

Abstract: Reproducibility of quantitative PCR results is dependent on the generation of consistent calibration curves via accurate volume transfers and instrument performance. A review of 14 standard curves, using two different QuantDuo^® standard DNA lots, showed variability of cycle threshold values between assays were larger than those of the Internal PCR Control (IPC). This prompted a set of experiments designed to determine the source of variability. Results showed that error introduced during DNA addition to the plate resulted in little variation. A comparison of seven independent series demonstrated cycle threshold variation between dilutions was larger than the variation expected from repeated samples. Modeling the influence of pipette errors on dilution series accuracy indicated that a more rigorous approach to external calibration curve production is required and showed that improvement in calibration curve stability is expected if the pipette conditions are carefully chosen and/or a single validated curve is utilized as the calibrator.     

Gamma Irradiation as a Biological Decontaminant and Its Effect on Common Fingermark Detection Techniques and DNA Profiling

Abstract:  The use of disease-causing organisms and their toxins against the civilian population has defined bioterrorism and opened forensic science up to the challenges of processing contaminated evidence. This study sought to determine the use of gamma irradiation as an effective biological decontaminant and its effect on the recovery of latent fingermarks from both porous and nonporous items. Test items were contaminated with viable spores marked with latent prints and then decontaminated using a cobalt 60 gamma irradiator. Fingermark detection was the focus with standard methods including 1,2-indanedione, ninhydrin, diazafluoren-9-one, and physical developer used during this study. DNA recovery using 20% Chelex extraction and quantitative real-time polymerase chain reaction was also explored. Gamma irradiation proved effective as a bacterial decontaminant with D -values ranging from 458 to 500 Gy for nonporous items and 797–808 Gy for porous ones. The results demonstrated the successful recovery of latent marks and DNA establishing gamma irradiation as a viable decontamination option.     

Study: The Lack of Significant Association of the Catechol-O-Methyl Transferase (COMT) Gene Polymorphism in Violent Offenders with Mental Retardation

Abstract:  Little is known about criminality of cognitively impaired people and also there have been no reports on the relationship between catechol- O -methyl transferase (COMT) and committed Mental Retardation (MR) subjects. In the present study, the association between committed (violent offences) MR subjects and genetic variants of COMT were investigated by using polymerase chain reaction and based restriction fragment length polymorphism methods. During 6 years of follow-up, 36 violent offenders with mild MR were investigated. Thirty-six control volunteers were included in the study as a control group. H/L polymorphism of the COMT gene was investigated in these two groups. In conclusion, the COMT gene genotype distribution and allele frequency is not significantly different between the two groups ( p  > 0.05). This result suggests that the H/L polymorphism of the COMT gene does not show an association with the potential of "commits-violent offense" of Turkish subjects with mental retardation, compared with control group.     

评鉴公司法之意思自治理念——以合同为视角

《公司法》作为一部张扬意思自治精神的法律被学界和实务界高度肯定。盛赞之后的思考是：意思自治既然是传统私法的灵魂和核心,那么对于在一定程度上被公法化了的公司法,意思自治是如何展开适用的,其本源性依据何在,其与合同法对于意思自治的诠释有什么不同以及其强制性规定在法律适用上是对意思自治的挟持还是力挺。基于以上法律困惑笔者展开以下分析：理论层面上,本文以合同为视角,以公司契约论为进路对意思自治制度在公司法背景下进行了寻根性研究;技术层面上,以《公司法》规范配置的设计为依托,深度挖掘我国公司法运用意思自治制度的积极价值和经验教训;操作层面上,从市场的深度融入、司法的谨慎介入以及规范的合理配置等角度提出了公司法关于意思自治制度的更新和完善。     

犯罪的标尺——浅议我国犯罪证明标准的相关问题

我国未来的犯罪证明标准应当是层次式和分解式相结合的体系,即以一个高度盖然的总体证明标准为顶端,在该顶端下分别以侦查、起诉和审判三阶段的完结条件为标志设立阶段性证明标准。对于总体证明标准,在将法条修正为:"证据事实清楚,证据材料确实、充分"的同时,必须不断完善刑事诉讼程序使之更为正当和合理,而且还应增强司法主体的证据意识;而在刑事诉讼各阶段,则根据该阶段的直接任务分别设立相应的阶段性证明标准。     

浅论家庭共有财产的认定

家庭共有财产属于家庭财产中的一个类型,该部分财产的准确认定对于正确解决家庭财产纠纷具有至关重要的意义。而不同的家庭财产有其不同特点,在认定标准上也不尽相同。在实践中,应根据不同财产产权的确认方式来对其进行认定,为正确解决家庭财产纠纷提供可操作性的标准。     

朱元璋倡廉惩贪措施的反思与借鉴

朱元璋大力反贪、促进廉政的一系列措施,其历史进步性是不容否定的。以史为鉴,当代反腐路径应是全社会齐动员,采取多种方法,多条途径,标本兼治,惩防并举。