Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs

McNevin, Dennis; Edson, Janette; Robertson, James; Austin, Jeremy J.

doi:10.1007/s12024-015-9679-3

Cited by 19 publications

(5 citation statements)

References 37 publications

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“…This result suggested that correct genotyping could be obtained when the PCR volume was reduced to 5 μl. In some forensic cases with trace or low levels of template DNA, the PCR reaction volume was often reduced to improve the reliability and sensitivity of STR profiling (Gibson‐Daw, Crenshaw, & McCord, 2018; McNevin, Edson, Robertson, & Austin, 2015). A lower PCR reaction volume would sometimes be chosen to deal with low copy DNA (Zhang et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

RETRACTED: Evaluation of a six‐dye multiplex composed of 27 markers for forensic analysis and databasing

Wang

Song

Xie

et al. 2020

Molec Gen & Gen Med

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Background Short tandem repeat (STR) markers play a significant role in genetic applications and have proved to be effective for the personal identification in forensic medicine. In this study, a six‐dye multiplex composed of 23 autosomal STR loci (TH01, D3S1358, Penta D, D6S1043, D21S11, TPOX, D1S1656, D12S391, Penta E, D10S1248, D22S1045, D19S433, D8S1179, D2S1338, D2S441, D18S51, vWA, FGA, D16S539, CSF1PO, D13S317, D5S818, D7S820), one Y chromosome STR (DYS391), two internal quality control markers (Quality Sensor QS1 and QS2), and Amelogenin was evaluated. Methods Evaluation studies, including PCR‐based studies, sensitivity studies, species specificity studies, stability studies, DNA mixture studies, concordance studies, and precision evaluations were performed according to the guidelines of “Validation Guidelines for Forensic DNA Analysis Methods (2016)” by the Scientific Working Group on DNA Analysis Methods (SWGDAM). In addition, the forensic characteristics of 357 unrelated male samples from Han and Hui populations in China were investigated using 27 markers. Results Full STR profiles were obtained from different reaction volumes (5 ~ 25 μl), cycle numbers (28 ~ 34 cycles) and annealing temperatures (58 ~ 62°C). All STR profiles were obtained at humic acid concentration of up to 200 ng/μl and hematin concentration of up to 500 μM. No peaks were observed in most common animal samples except two innovative internal PCR controls (Quality Sensor QS1 and QS2). The six‐dye multiplex showed a notably high value for the combined probability of exclusion (CPE), exhibiting values of with 0.99999999977688 in the Han population and 0.999999999583875 in the Hui population. The values of combined probability of discrimination (CPD) were 0.999999999999999999999999999997453 in the Han population and 0. 999999999999999999999999999994398 in the Hui population. In addition, concordance studies showed that there was no difference with the AGCU Express Marker 22 Kit. Conclusion The results indicated that the Investigator® 26plex QS Kit is a robust, reliable, and suitable tool for forensic analysis and databasing.

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Section: Resultsmentioning

confidence: 99%

RETRACTED: Evaluation of a six‐dye multiplex composed of 27 markers for forensic analysis and databasing

Wang

Song

Xie

et al. 2020

Molec Gen & Gen Med

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“…We observed that the incorporation of multiple TaqMan probes for signal generation did somewhat reduce overall PCR efficiency, necessitating the use of a higher number of cycles to reach the PCR plateau phase. However, we note that overall assay time could likely be reduced though optimization of the PCR reaction (e.g., increasing the concentration of DNA polymerase or utilizing a DNA polymerase with stronger exonuclease activity). While LiNC PCR can be performed using conventional TaqMan probes, incorporation of LNA sequences allows for the use of shorter, more specific probes, thereby increasing the possible number of probe sites in each LP as well as improving the performance of the method by enhancing the signal-to-noise ratio. , It is worth noting that the LiNC PCR method demonstrated here for broad-based detection of target sequences; it is in principle readily amenable and ideally suited for use with digital PCR (dPCR)-based methods , to unlock the full multiplexing potential of the technique.…”

Section: Discussionmentioning

confidence: 99%

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection

et al. 2021

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Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays. The technique relies upon a simple, preliminary ligation reaction in which target DNA sequences are converted to PCR template molecules with distinct endpoint fluorescence signatures. Universal TaqMan probes are used to create target-specific multicolor fluorescence signals that can be readily decoded to identify amplified targets of interest. We demonstrate the LiNC PCR technique by implementing a two-color-based assay for detection of 10 ovarian cancer epigenetic biomarkers at analytical sensitivities as low as 60 template molecules with no detectable target cross-talk. Overall, LiNC PCR provides a simple and inexpensive method for achieving high-dimensional multiplexing that can be implemented in manifold molecular diagnostic applications.

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“…Amplification kits are generally not considered to be a significant factor in a profiles' dropout rate, however many studies have compared the performance of different STR kits and also at low DNA amounts [24,40,41], i.e., for a comprehensive investigation of STR amplification success for touch DNA applications [12]. Moreover, a plethora of studies [24,27,[42][43][44][45][46][47][48] indicate that the use of a reduced amplification volume increases PCR sensitivity. Tay et al [40] examined the performance of three forensic autosomal STR kits on buccal cells according to their manufacturers' recommendations (using DNA ranges from 0.008 to 2 ng).…”

Section: Dilution Studymentioning

confidence: 99%

A systematic approach to improve downstream single‐cell analysis for the DEPArray™ technology

Schulte

Marciano

Scheurer

et al. 2023

Journal of Forensic Sciences

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Most commercially available STR amplification kits have never been fully validated for low template DNA analysis, highlighting the need for testing different PCR kits and conditions for improving single‐cell profiling. Here, current strategies rely mainly on adjusting PCR cycle number and analytical threshold settings, with a strong preference for using 30 amplification cycles and thresholds at 30–150 RFU for allele detection. This study aimed to (1) determine appropriate conditions for obtaining informative profiles utilizing a dilution series, and (2) test the outcome on single cells using the DEPArray™ technology. Four routinely applied forensic STR kits were compared by using three different amplification volumes and DNA dilutions down to 3.0 pg, while two well‐performing kits were used for single/pooled leucocyte and sperm cell genotyping. Besides reduced costs, the results demonstrate that a 50%–75% PCR volume reduction was beneficial for peak height evaluation. However, this was counteracted by an increased artifact generation in diluted DNA volumes. Regarding profile completeness, the advantage of volume reduction was only prominent in samples processed with Fusion 6C. For single and pooled cells, ESIFast and NGMDetect provided a solid basis for consensus profiling regarding locus failure, although locus dropouts were generally observed as stochastic events. Amplification volume of 12.5 μL was confirmed as appropriate in terms of peak heights and stutter frequencies, with increased stutter peaks being the main artifact in single‐cell profiles. Limitations associated with these analyses are discussed, providing a solid foundation for further studies on low template DNA.

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Reduced reaction volumes and increased Taq DNA polymerase concentration improve STR profiling outcomes from a real-world low template DNA source: telogen hairs

Abstract: Overall, this study highlights the importance and benefit of optimizing PCR conditions and developing improved laboratory methods to amplify and analyze LTDNA.

Cited by 19 publications

References 37 publications

RETRACTED: Evaluation of a six‐dye multiplex composed of 27 markers for forensic analysis and databasing

RETRACTED: Evaluation of a six‐dye multiplex composed of 27 markers for forensic analysis and databasing

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection

A systematic approach to improve downstream single‐cell analysis for the DEPArray™ technology

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