Statistics for analytical chemistry

In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance.

A Study of PCR Inhibition Mechanisms Using Real Time PCR

Noninvasive genetic approaches continue to improve studies in molecular ecology, conservation genetics and related disciplines such as forensics and epidemiology. Noninvasive sampling allows genetic studies without disturbing or even seeing the target individuals. Although noninvasive genetic sampling has been used for wildlife studies since the 1990s, technological advances continue to make noninvasive approaches among the most used and rapidly advancing areas in genetics. Here, we review recent advances in noninvasive genetics and how they allow us to address important research and management questions thanks to improved techniques for DNA extraction, preservation, amplification and data analysis. We show that many advances come from the fields of forensics, human health and domestic animal health science, and suggest that molecular ecologists explore literature from these fields. Finally, we discuss how the combination of advances in each step of a noninvasive genetics study, along with fruitful areas for future research, will continually increase the power and role of noninvasive genetics in molecular ecology and conservation genetics.

https://www.fs.fed.us/rm/pubs_other/rmrs_2009_beja_pereira_a001.pdf

Advancing ecological understandings through technological transformations in noninvasive genetics.

Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs.

The ISFG membership consists of scientists and medical professionals specialized in using genetic testing for kinship analysis and the individualization of biological material. This expertise makes the forensic geneticist a resource of advice to international and national organizations dealing with human identifications and causes many DNA laboratories to get involved in DVI tasks. The present recommendations are meant to educate more forensic geneticists about their potential involvement in mass fatality preparedness and possible DVI efforts, as well as to provide practical guidance for each of the laboratories’ individual tasks. The idea to work on DNA-specific recommendations was born after a round table discussion dealing with the 2004 Tsunami disaster in south east Asia during the 21st congress of the International Society for Forensic Genetics on the Azores, Portugal, in September 2005. The ensuing discussion between scientists and pathologists that had been involved in the International Center in Khao Lak, Thailand, revealed the need for the scientific community to be better prepared to answer the local authorities’ questions by formulating generally acceptable scientific standards for the most efficient use of DNA-based victim identification methods. These recommendations, as well as the many cited references, are intended to provide guidance on establishing preparedness for the forensic genetics laboratory, on collecting and storing ante-mortem and post-mortem samples suitable for DNA analysis, on DNA extraction and genetic typing strategies, on data management, and on issues related to the biostatistical interpretation and reporting of results. # 2006 Elsevier Ireland Ltd. All rights reserved.

/pdf/dna-commission-of-the-international-society-for-forensic-2fwhdant4j.pdf

DNA Commission of the International Society for Forensic Genetics (ISFG): Recommendations regarding the role of forensic genetics for disaster victim identification (DVI)

In forensic DNA analysis, the samples recovered from the crime scene are often highly degraded leading to poor PCR amplification of the larger sized STR loci. To avoid this problem, we have developed STR markers with redesigned primer sequences called "Miniplexes" to produce smaller amplicons. To assess the effectiveness of these kits, we have tested these primer sets with enzymatically degraded DNA and compared the amplifications to a commercial kit. We also conducted sensitivity and peak balance studies of three Miniplex sets. Lastly, we report a case study on two human skeletal remain samples collected from different environmental conditions. In both types of degraded DNA, the Miniplex primer sets were capable of producing more complete profiles when compared to the larger sized amplicons from the commercial kit. Correct genotypes were obtained at template concentrations as low as 31 pg/25 цL. Overall, our data confirm that our redesigned primers can increase the probability of obtaining a usable profile in situations where standard kits fail.

https://projects.nfstc.org/workshops/resources/literature/A%20Study%20on%20the%20Effects%20of%20Degradation%20and.pdf

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets.

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex s 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Pow- erplex s 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains.

TABLE 1—Summary of 15 discordant STR profiling results observed in this study between the Identifiler kit and our Miniplex assays for 12 different African American (AA) and 3 Hispanic (H) samples. PowerPlex 16 (PP16) results all agree with the Identifiler results for these 15 samples. Sigle allele shifts of 1 repeat in the D13S317 heterozygotes are likely due to a 4 base pair deletion in the flanking region outside of the Miniplex primer binding site (see Ref 2). Allele dropout at D5S818 (see Ref 2), D13S317, and vWA (see Ref 3) are likely due to primer binding site mutations at specific alleles.

Concordance study between Miniplex assays and a commercial STR typing kit.

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.

/pdf/developmental-validation-of-reduced-size-str-miniplex-primer-15lii7jbtg.pdf

Denise T. Chung

Papers

A study on the effects of degradation and template concentration on the amplification efficiency of the STR Miniplex primer sets.

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains.

Concordance study between Miniplex assays and a commercial STR typing kit.

Developmental validation of reduced-size STR Miniplex primer sets.