Removal of a PCR Inhibitor and Resolution of DNA STR Types in Mixed Human-Canine Stains from a Five Year Old Case

Shutler, Gary; Gagnon, Pierre; Verret, Gary; Kalyn, Howard; Korkosh, Sandra; Johnston, Eric V.; Halverson, Joy

doi:10.1520/jfs14520j

Cited by 60 publications

(40 citation statements)

References 12 publications

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“…Robust interpretation of DNA profiles, including the detection of PCR inhibition, is vital for the interpretation of DNA evidence and can have a significant impact on the outcome of criminal cases [6,7]. Various approaches have been developed to detect PCR inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

et al. 2011

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DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.

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Section: Introductionmentioning

confidence: 99%

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

et al. 2011

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“…However, the success rate of diagnostic PCR analysis is lowered by the presence of PCRinhibitory substances [2], a problem especially prominent in forensic DNA analysis due to the often complex sampling environment at crime scenes [3][4][5]. Recently, we reported that 12% of volume crime saliva stains with adequate DNA amounts analyzed at our laboratory still produced negative DNA profiles due to the presence of inhibitors [6].…”

Section: Introductionmentioning

confidence: 99%

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Hedman

Nordgaard

Dufva

et al. 2010

Analytical Biochemistry

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a b s t r a c tThe success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 ''inhibited" crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.

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“…Seventy-two million pet dogs are estimated to live in the USA (3), and because many dog owners live in close proximity to their pets, canine DNA evidence may be associated with crimes. Shed dog hairs can be transferred among individuals involved in a crime and can link a suspect to a crime scene, a suspect to a victim, or a victim to a crime scene (4,5). Dog evidence can also be used in dog attack cases and abuse cases (6).…”

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confidence: 99%

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

Dayton

Koskinen²,

Tom

et al. 2009

Croat Med J

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Aim To develop a reagent kit that enables multiplex polymerase chain reaction (PCR) amplification of 18 short tandem repeats (STR) and the canine sex-determining Zinc Finger marker.Methods Validation studies to determine the robustness and reliability in forensic DNA typing of this multiplex assay included sensitivity testing, reproducibility studies, intra-and inter-locus color balance studies, annealing temperature and cycle number studies, peak height ratio determination, characterization of artifacts such as stutter percentages and dye blobs, mixture analyses, species-specificity, case type samples analyses and population studies. ResultsThe kit robustly amplified domesticated dog samples and consistently generated full 19-locus profiles from as little as 125 pg of dog DNA. In addition, wolf DNA samples could be analyzed with the kit. ConclusionThe kit, which produces robust, reliable, and reproducible results, will be made available for the forensic research community after modifications based on this study's evaluation to comply with the quality standards expected for forensic casework.

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Removal of a PCR Inhibitor and Resolution of DNA STR Types in Mixed Human-Canine Stains from a Five Year Old Case

Cited by 60 publications

References 12 publications

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

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Removal of a PCR Inhibitor and Resolution of DNA STR Types in Mixed Human-Canine Stains from a Five Year Old Case

Cited by 60 publications

References 12 publications

Development of internal amplification controls for DNA profiling with the AmpFℓSTR® SGM Plus® kit

Development of internal amplification controls for DNA profiling with the AmpFℓSTR® SGM Plus® kit

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis

Developmental Validation of Short Tandem Repeat Reagent Kit for Forensic DNA Profiling of Canine Biological Materials

Contact Info

Product

Resources

About

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit

Development of internal amplification controls for DNA profiling with the AmpFℓSTR^® SGM Plus^® kit