A real-time multiplex SNP melting assay to discriminate individuals

A method that quickly and inexpensively differentiates crime scene samples from multiple donors would expedite casework analysis by allowing the selection of probative items requiring comprehensive testing. This new method need not be perfectly definitive nor give a complete 13 locus short tandem repeat (STR) profile; it simply must be able to differentiate between most victim and suspect samples. We describe the development of multiplex, single nucleotide polymorphism (SNP), fluorescence resonance energy transfer-based real-time polymerase chain reaction (PCR) assays to fulfill this need. Dual probes, one fluorescently labeled and the other labeled with a quencher, are monitored during a melt analysis to reveal an increase in fluorescence, which allows the assessment of the two SNP alleles. Two alternate 6-plex assays (with and without gender determination) have been developed for the six-color RG6000 real-time instrument (Corbett Robotics, Inc.) and one seven SNP plus gender assay (performed as two 4-plex assays, one with gender the other without) have been developed for use in four/five color real-time instruments. This technique can discriminate between 95% and 99% of samples from different individuals. This assay is fast (approximately 2 h), much less expensive than STR analysis, and uses a real-time PCR instrument which is found in most forensic and molecular biology labs.     

Intimate Partner Violence and Risk for Child Neglect during Early Childhood in a Community Sample of Fragile Families

The current study explores the relationship between child neglect and intimate partner violence (IPV) in a longitudinal community sample of 1,740 families with young children, with a special focus on the association between specific typologies of both neglect behaviors and IPV. We focused on families followed across early childhood, because infants and toddlers are at the greatest risk of exposure to neglect (the most prevalent type of child maltreatment), and this period spanning the transition to parenthood presents heightened risk for IPV. We found evidence that coercive IPV is an important driver of the connections between IPV and subsequent neglect through affecting the mother’s well-being and ability to provide basic care and nurturance. Implications for intervention and future work addressing definitions and pathways to neglect are discussed.     

The Shanghai Cooperation Organization,trade, and the roles of Iran,India and Pakistan

This article seeks to explore the implications of Shanghai Cooperation Organization's (SCO) engagement with India, Pakistan and Iran. Not in terms of power-politics or as a counterbalance to the USA as this has been explored elsewhere, but what practical problems such an expanded organization could help solve, what opportunities it could realize, and how SCO's engagement in trade is a function of favourable political and bilateral developments in the region. It is argued here that the trade, infrastructure and energy sectors are of particular importance and that substantial potential gains could be realized if coordination is improved. Nevertheless, it is also recognized that China, Russia, Pakistan, India and Iran may have lower standards of democratic development and economic transparency than the West. What is the motivation behind the SCO's engagement with India, Pakistan and Iran? Should this engagement be conceived only in terms of balancing US unipolarity or are there legitimate concerns of increasing regional cooperation in Eurasia?     

An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples

The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).     

Development of a real-time method to detect DNA degradation in forensic samples

Abstract: Knowledge of the degradation state of evidentiary DNA samples would allow selection of the appropriate analysis method (standard short tandem repeats [STRs] vs. mini STRs vs. mtDNA). This article describes the development of a Plexor® technology/real‐time PCR DNA degradation detection assay, which uses a common forward primer and two reverse primers (different fluorophores) to generate two Alu amplicons (63 and 246 bp). This very sensitive assay was optimized for reaction volume, cycle number, anneal/extend time, and temperature. Using DNA samples degraded with DNaseI, the ratio of the concentration of the short amplicon to the concentration of the long amplicon (degradation ratio) was increased versus time of degradation. Experiments were performed on a variety of environmentally degraded samples (age, sunlight, heat) and with seven commonly encountered forensic inhibitors. The degradation ratio was found to predict the observed loss of larger STR loci seen in the analysis of comprised samples.     

Development of an Alu-based,QSY 7-labeled primer PCR method for quantitation of human DNA in forensic samples

Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive. This paper describes the development of a new technique based on PCR amplification of a repetitive Alu sequence. Specific primers were used to amplify a 124-bp fragment of Alu sequence; amplification was detected by SYBR Green I staining in a fluorescent plate reader. To reduce background in the plate reader assay, QSY-7 labeled primers were utilized. The assay was tested on animal DNAs, human blood spots, mock crime samples, and degraded DNA in comparison with the slot blot technique. The QSY Alu assay has a dynamic range of 10 ng to 10 pg, and is sensitive, specific, fast, quantitative, and comparable in cost to the slot blot assay.     

Simultaneous determination of total human and male DNA using a duplex real-time PCR assay

A single duplex assay to determine both the amount of total human DNA and the amount of male DNA in a forensic sample has been developed. This assay is based on TaqMan technology and uses the multicopy Alu sequence to quantitate total human DNA and the multicopy DYZ5 sequence to quantitate Y chromosomal (male) DNA. The assay accepts a wide concentration range of input DNA (2 muL of 64 ng/microL to 0.5 pg/microL), and also allows detection of PCR failure. The PCR product sizes Alu (127 bp) and DYZ5 (137bp) approximate that of the smaller short tandem repeats (STRs) which should make the assay predictive of STR success with degraded DNA. The assay was optimized for probe/primer concentrations and BSA addition and validated on its reproducibility, on its human specificity, on its nonethnic variability, for artificial mixtures and adjudicated casework, for the effect of inhibitors and for state of DNA degradation. This assay should prove very usual in forensic analyses because knowing the relative amounts of male versus female DNA can allow the examiner to decide which samples may yield the most probative value in a case or direct the samples to methods that would yield the greatest information.