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Bronislav Horns
Bronislav Horns

Forensic Dna Typing Protocols Pdf Download

This volume presents a series of protocols and methods, some of which are not widely used by researchers/practitioners, and will aid in the execution of different laboratory techniques. Forensic DNA Typing Protocols, Second Edition is arranged into a series of related chapters. Chapter 1-3 examines two different aspects of RNA analysis for body fluid identification. Chapters 4-7 focuses on the storage of biological materials and the extraction of DNA from hard tissues. Chapters 8-10 present methods for monitoring the quality of DNA extracts, and steps to aid in the purification of DNA. Chapters 11-16 talk about methods on non-standard markers, such as INDELs, Y chromosome STRs, and mitochondrial DNA. Detailed procedures and data analysis for phenotypes and ancestry are explored in Chapter 17-19. The last chapter (20) looks at the application of DNA typing to the identification of non-human material to species level. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Practical and thorough, Forensic DNA Typing Protocols, Second Edition, is a valuable resource for forensic specialists, researchers, and anyone interested in the field of forensic science.

forensic dna typing protocols pdf download

The advantages of the CPI approach are thought to be its simplicity and the fact that the number of contributors need not be assumed in the calculation. However, even with simplicity, recently, in the U.S., interpretation protocols used for DNA mixtures using the CPI method have been criticized when applied to forensic mixtures for which it is not suited, highlighting issues of effective communication and technology transfer to the end users of the forensic science community [14]. One should be wary of deceptively simple solutions to complex problems as it is possible that the perceived simplicity of the CPI statistic has led in some instances to incorrect applications of the approach. While the number of alleles is used to generate a CPI statistical estimate, it is incumbent upon the user to evaluate a mixture based on the possible genotypes of the contributors and to consider the potential of missing data (i.e., allele drop-out) based on peak height observations at other loci in the profile and the possibility of allele stacking.

Given emerging criticism of methods used in forensic DNA mixture analysis, interpretation and statistical evaluation - particularly in the U.S. - it is timely to revisit and reinforce the foundational principles of interpretation of mixtures and subsequent computation as it relates to the CPI (or CPE). The authors recognize and advocate the community as a whole move towards the use of probabilistic genotyping methods [9, 17, 22, 23] with proper validation. However, in the interim, it has become evident that a specific CPI protocol is needed to guide practitioners who currently use it and for re-analysis of past cases in which use of the CPI method may not have considered the guidelines detailed herein. All methods, including probabilistic genotyping and the CPI-based approach, require the ability to deconvolve mixtures.

Although the establishment of a consensus set of core STR loci allows comparisons of profiles across jurisdictions and over time through use of national databases, it may also be simultaneously stifling opportunities for the improvement in the quality and efficiency of the service provided. Changing the type of markers used, from STRs to single nucleotide polymorphisms (SNPs), may result in increased success from more forensic samples and be more adaptable to high-throughput and/or miniaturized typing systems. Theoretically, the smaller amplicon sizes of SNPs lend themselves well to the production of genetic profiles from both degraded and trace DNA. Their reduced level of polymorphism relative to the routinely used STRs is, however, a disadvantage. With sufficient numbers this can be overcome, although it may make mixture resolution more difficult. Whilst sensitive SNP-based individualization profiling systems are available [25, 26] they are not routinely used.

Surveys of forensic practitioners regarding aspects of training, proficiency testing, procedures, methods, policies, contamination prevention, data collection and communication relating to forensic trace DNA have highlighted the need for improvements in these areas [135, 136]. A number of recent reports have recommended the need for substantially greater investment into forensic services related research and development [82, 219]. This review identifies how far we have come in the use of trace DNA in order to assist forensic investigations in recent years, but it also identifies several opportunities for improvement in most facets of trace DNA work. A deeper consideration of workflow processes and priorities may yield alternative protocols that allow the use of a greater portion of the available DNA, with greater sensitivity, thus increasing the chance of generating fuller and easier to interpret profiles. Further research will improve the utilisation and benefits of collecting and typing trace DNA in forensic investigations.

Abstract:The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.Keywords: degraded DNA; massively parallel sequencing (MPS); mitochondrial DNA; forensic DNA profiling; ancient DNA; human identification

Assessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms.

We recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles.

The object of forensic DNA analysis is to generate individual-specific DNA profiles from crime scene stains and reference samples, thereby linking perpetrators to crimes. The analytical process includes sampling, DNA extraction/purification, and amplification of certain genetic markers (Short Tandem Repeats, STR) using the polymerase chain reaction (PCR). The actual DNA profile is generated by capillary electrophoresis separation of DNA fragments and detection using fluorescently labeled primers. An electropherogram (EPG) is produced where the intensity of the allelic peaks corresponds to the amount of produced DNA fragments, and the balance between peaks gives information on the reliability of the DNA profile (Figure 1). The amount and purity of the DNA is determined by all steps in the analytical process and subsequently affect the quality of the EPG/DNA profile. Consequently, assessment of DNA profile quality is vital both for establishing the evidentiary value of a certain DNA profile and for comparing the relative performance of different DNA analysis protocols, e.g., in validation studies.

We recently developed the forensic DNA profile index (FI), a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles [15]. FI combines intensity and balance into one single, easily interpretable numerical index. FI is constructed by using Principal Components Analysis (PCA) on the following DNA profile quality measures: total allelic peak height (intensity), balance between allelic peaks within heterozygous loci (intra-locus or local balance), and balance between STR markers (inter-loci or global balance). The ranking index is based on empirical data taking into account statistical properties of such data as well as common opinions about what is considered a high or low quality EPG/DNA profile. Here we present the construction of FI, describing the applied mathematical and statistical methodologies. We show how to adapt the ranking index for any STR-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles. 350c69d7ab


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