Human genomic DNA quantitation system, H-Quant: Development and validation for use in forensic casework

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR® Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR® Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/μL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories. Copyright © 2006 by American Academy of Forensic Sciences

Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues

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

ABSTRACT: The PrepFiler TM 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 lL 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 Ò 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. KEYWORDS: forensic science, DNA extraction, DNA purification, DNA isolation, DNA typing, short tandem repeat profiling Profiling for short tandem repeat (STR) loci is routinely performed in forensic, DNA database and paternity applications for human identification, and lineage studies (1,2). The genotyping protocol is comprised of extraction of DNA from the biological sample, quantification of the DNA, amplification of STR loci, separation of amplified products using gel or capillary electrophoresis, and analysis of the results. The past two decades have witnessed advancements in the development of new technologies for STR analysis. These advancements include development of real-time polymerase chain reaction (PCR) methods for quantification of human DNA (3-7), multiplex STR kits for profiling of autosomal and Y-STRs (8-14), capillary electrophoresis instruments (15,16), fluorescence imaging systems (17), and data analysis programs Forensic analysts come across a variety of biological samples including stains of blood, saliva or semen on different substrates, swabs of body surface, hair, bones, and finger nail scrapings that are exposed to a range of environmental insults. DNA in the cells is associated with a number of physiological components and other macromolecules that protect the DNA in vivo. If these substances are not removed during the DNA extraction procedure, they can interfere in the downstream processes of DNA analysis such as PCR. Therefore, it is important that the procedure used for preparing the DNA is efficient and extracts the DNA in a highly purified form. A wide variety of methods based on different principles are available for extraction of DNA (2,22-26). These include Chelex Ò (Sigma-Aldrich, St. Louis, MO) extraction, phenol-chloroform, silica membranes, silica-coated magnetic beads, ion exchange membranes, and magnetic beads with an ionic surface. Kishore et al. (27) have proposed that silica-coated particles bind a certain portion of DNA in the sample via nonspecific adhesion providing low yields of DNA from samples containing smaller quantities of biological material. The principle of DNA extraction using phenol is mainly based upon denaturation of the contaminants including proteins and other macromolecules and isolation of DNA in purified form. It is important to note that the traditional phenol-chloroform organic extraction method, often referred to as gold standard, is still a predominant method for DNA isolation from forensic casework samples. Isolation of DNA from forensic evidence samples, therefore, can be challenging and create bottlenecks in the sample processing workflow. The quality of DNA extract is of utmost importance as the ultimate goal of DNA analysis is to obtain an STR profile devoid of any PCR artifacts. In general, it is desirable for a forensic analyst to have an extraction methodology that enables: (i) the isolation of DNA from biological samples that contain small quantities of biological material; (ii) obtaining the DNA at a high concentration so that the volume of extract used for PCR is minimal; (iii) the removal of PCR inhibitors or substances that interfere with the PCR; (iv) the extraction of DNA from a variety of biological samples; and (v) the adaptation of the manual protocol and its chemistry to automation. We describe an innovative method, the PrepFiler TM Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA), that meets all of these criteria. The developed method enables the isolation of genomic DNA from forensic biological samples that is free of PCR inhibitors and ready for downstream applications such as real-time PCR and genotyping. Materials and Methods Biological samples such as blood, saliva, and semen were obtained from Serological Research Institute (Richmond, CA)