This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Section Editor and Authors after a thorough investigation. While NIST informed the journal that it followed appropriate human subjects protocols, it has come to light that the manuscript fails to indicate whether consent was obtained for all sample acquisition. The article further incorrectly implies that NIST collaborated with the Guangzhou Forensic Science Institute. All authors have been informed of this decision. Apologies are offered to readers of the journal that this was not detected during the submission process.

Microsatellite instability (MSI) is an evolving biomarker for cancer detection and treatment. MSI was first used to identify patients with Lynch syndrome, a hereditary form of colorectal cancer (CRC), but has recently become indispensable in predicting patient response to immunotherapy. To address the need for pan-cancer MSI detection, a new multiplex assay was developed that uses novel long mononucleotide repeat (LMR) markers to improve sensitivity. A total of 469 tumor samples from 20 different cancer types, including 319 from patients with Lynch syndrome, were tested for MSI using the new LMR MSI Analysis System. Results were validated by using deficient mismatch repair (dMMR) status according to immunohistochemistry as the reference standard and compared versus the Promega pentaplex MSI panel. The sensitivity of the LMR panel for detection of dMMR status by immunohistochemistry was 99% for CRC and 96% for non-CRC. The overall percent agreement between the LMR and Promega pentaplex panels was 99% for CRC and 89% for non-CRC tumors. An increased number of unstable markers and the larger size shifts observed in dMMR tumors using the LMR panel increased confidence in MSI determinations. The LMR MSI Analysis System expands the spectrum of cancer types in which MSI can be accurately detected.

Purpose Estimating cancer risk associated with interplanetary space travel is complicated. Human exposure data to high atomic number, high-energy (HZE) radiation is lacking, so data from low linear energy transfer (low-LET) gamma-ray radiation is used in risk models, with the assumption that HZE and gamma-ray radiation have comparable biological effects. This assumption has been challenged by reports indicating that HZE radiation might produce more aggressive tumors. The goal of this research is to test whether high-LET HZE radiation induced tumors are more aggressive. Materials and methods Murine models of mammary and liver cancer were used to compare the impact of exposure to 0.2Gy of 300MeV/n silicon ions, 3 Gy of gamma-rays or no radiation. Numerous measures of tumor aggressiveness were assessed. Results For the mammary cancer models, there was no significant change in the tumor latency or metastasis in silicon-irradiated mice compared to controls. For the liver cancer models, we observed an increase in tumor incidence but not tumor aggressiveness in irradiated mice. Conclusion Tumors in the HZE-irradiated mice were not more aggressive than those arising from exposure to low-LET gamma-rays or spontaneously. Thus, enhanced aggressiveness does not appear to be a uniform characteristic of all tumors in HZE-irradiated animals.

The VersaPlex™ 27PY System is an STR multiplex from Promega offering co-amplification of 27 loci. This six-dye multiplex was designed for casework samples and includes loci that meet database requirements for the US (CODIS), Europe (ESS) and China (Chinese National Database). The 23 autosomal loci include the D6S1043 locus because of its high level of discrimination power, especially in Asian populations. The multiplex also includes two rapidly-mutating Y-STRs that can provide useful genetic information for forensic samples. In this article we describe the developmental validation experiments performed on the VersaPlex™ 27PY System that were designed to meet SWGDAM requirements. Changes in the performance of the multiplex are documented in response to reaction variables including PCR inhibitors, reaction volume, cycle number, annealing temperature, and magnesium concentration. The sensitivity, specificity, precision, and reproducibility of the VersaPlex™ 27PY System were also evaluated. The results of the experiments demonstrate the capabilities of the VersaPlex™ 27PY System as a tool for forensic laboratories.

Research in toxicology relies on in vitro models such as cell lines. These living models are prone to change and may be described in publications with insufficient information or quality control testing. This article sets out recommendations to improve the reliability of cell-based research.

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research.

Background: A new multiplexed biomarker panel is being developed for detection of microsatellite instability (MSI) that is more sensitive than currently available systems. Preliminary research data shows increased MSI sensitivity for colon polyps and endometrial (EC), skin and prostate cancers. The sensitivity of this Pan-Cancer MSI System is being further verified on 14 different cancer types. Methods: Selection of the new microsatellite biomarkers was done by screening 160 patients ≤55 years with ≥1 polyp and 100 EC patients ≤ 50 years for MSI. The expanded study uses samples from 100 Lynch syndrome colorectal cancers (CRC), 100 sporadic MSI-High CRC, 100 sporadic MSI stable CRC and 219 extra-colonic cancers obtained from the Colon Cancer Family Registry. DNA samples are being tested for MSI using two pan-cancer systems: Promega’s MSI Analysis System version 1.2 and the improved prototype Pan-Cancer MSI System. Mutations in mismatch repair (MMR) and BRAF genes were tested, as well as MMR expression by IHC. Results: 2.3% of colon polyps were MSI-High for the MSI Analysis System compared to 5.4% with the new prototype Pan-Cancer MSI System. Sensitivity and specificity of the new biomarker panel for detection of MMR deficient lesions was 100% and 96%. Similarly, sensitivity of the new biomarker panel for EC was about 2-fold higher. Allele size changes for MSI-High samples were significantly larger with the new biomarkers making MSI classification highly accurate and robost. The MSI and IHC results were highly correlated. Evaluation of the new biomarker panel is being performed on over 500 cancer samples from 14 different cancer types. Conclusions: Research results indicate that MSI sensitivity for colonic polyps and many extra-colonic cancers can be increased by at least 2-fold over current MSI systems using the new MSI biomarker panel. The improved sensitivity of the Pan-Cancer MSI System should improve detection of MSI in an expanded number of cancer types and facilitate identification of individuals with both sporadic and hereditary MSI-High cancers. Legal entity responsible for the study: Promega Corporation. Funding: Promega Corporation. Disclosure: J. Bacher: Employee: Promega Corporation. R. Halberg, P. Ward, K. Murphy, J. Eshleman: Corporate sponsored research funds: Promega. E. Udho, M. Uhr: Employee: Promega. D. Storts: Employee and stock ownership: Promega. All other authors have declared no conflicts of interest.

Forensic DNA analysis requires several steps, including DNA extraction, PCR amplification, and separation of PCR fragments. Intuitively, there are numerous situations where it would be beneficial to speed up the overall DNA analysis process; in this work, we focus on the most time-consuming component in the analysis pipeline, namely the polymerase chain reaction (PCR). Primers were specially designed to target 10 human genomic loci, all yielding amplicons shorter than 350 bases, for ease of downstream integration with on-board microchip electrophoresis. Primer concentrations were adjusted specifically for microdevice amplification, resulting in well-balanced short tandem repeat (STR) profiles. Furthermore, studies were performed to push the limits of the DNA polymerase to achieve rapid, multiplexed PCR on various substrates, including transparent and black polyethylene terephthalate (Pe), and with two distinct adhesives, toner and heat sensitive adhesive (HSA). Rapid STR-based multiplexed PCR amplification is demonstrated in 15 min on a Pe microdevice using a custom-built system for fluid flow control and thermocycling for the full 10-plex, and in 10 min for a smaller multiplex consisting of six core CODIS loci plus Amelogenin with amplicons shorter than 200bp. Lastly, preliminary studies indicate the capability of this PCR microdevice platform to be integrated with both upstream DNA extraction, and downstream microchip electrophoresis. This, coupled to the use of reagents that are compatible with lyophilization (lyo-compatible) for PCR, represents the potential for a fully integrated rotationally-driven microdevice for complete forensic DNA analysis.

Current forensic DNA analysis predominantly involves identification of human donors by analysis of short tandem repeats (STRs) using Capillary Electrophoresis (CE). Recent developments in Massively Parallel Sequencing (MPS) technologies offer new possibilities in analysis of STRs since they might overcome some of the limitations of CE analysis. In this study 17 STRs and Amelogenin were sequenced in high coverage using a prototype version of the Promega PowerSeq™ system for 297 population samples from the Netherlands, Nepal, Bhutan and Central African Pygmies. In addition, 45 two-person mixtures with different minor contributions down to 1% were analysed to investigate the performance of this system for mixed samples. Regarding fragment length, complete concordance between the MPS and CE-based data was found, marking the reliability of MPS PowerSeq™ system. As expected, MPS presented a broader allele range and higher power of discrimination and exclusion rate. The high coverage sequencing data were used to determine stutter characteristics for all loci and stutter ratios were compared to CE data. The separation of alleles with the same length but exhibiting different stutter ratios lowers the overall variation in stutter ratio and helps in differentiation of stutters from genuine alleles in mixed samples. All alleles of the minor contributors were detected in the sequence reads even for the 1% contributions, but analysis of mixtures below 5% without prior information of the mixture ratio is complicated by PCR and sequencing artefacts.

Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant (R) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant (R) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation. (C) 2016 The Author(s). Published by Elsevier Ireland Ltd. All rights reserved.

We demonstrate the capabilities of a centrifugal polyethylene terephthalate toner (PeT) microdevice for genetic analysis of short tandem repeats (STR) via PCR amplification.

The PowerPlex(®) Fusion 6C System is a 27-locus, six-dye, multiplex that includes all markers in the expanded CODIS core loci and increases overlap with STR database standards throughout the world. Additionally, it contains two, rapidly mutating, Y-STRs and is capable of both casework and database workflows, including direct amplification. A multi-laboratory developmental validation study was performed on the PowerPlex(®) Fusion 6C System. Here, we report the results of that study which followed SWGDAM guidelines and includes data for: species specificity, sensitivity, stability, precision, reproducibility and repeatability, case-type samples, concordance, stutter, DNA mixtures, and PCR-based procedures. Where appropriate we report data from both extracted DNA samples and direct amplification samples from various substrates and collection devices. Samples from all studies were separated on both Applied Biosystems 3500 series and 6-dye capable 3130 series Genetic Analyzers and data is reported for each. Together, the data validate the design and demonstrate the performance of the PowerPlex(®) Fusion 6C System.

Microsatellite instability (MSI) occurs in over 90% of Lynch syndrome cancers and is considered a hallmark of the disease. MSI is an early event in colon tumor development, but screening polyps for MSI remains controversial because of reduced sensitivity compared to more advanced neoplasms. To increase sensitivity, we investigated the use of a novel type of marker consisting of long mononucleotide repeat (LMR) tracts. Adenomas from 160 patients, ranging in age from 29-55 years old, were screened for MSI using the new markers and compared with current marker panels and immunohistochemistry standards. Overall, 15 tumors were scored as MSI-High using the LMRs compared to 9 for the NCI panel and 8 for the MSI Analysis System (Promega). This difference represents at least a 1.7-fold increase in detection of MSI-High lesions over currently available markers. Moreover, the number of MSI-positive markers per sample and the size of allelic changes were significantly greater with the LMRs (p = 0.001), which increased confidence in MSI classification. The overall sensitivity and specificity of the LMR panel for detection of mismatch repair deficient lesions were 100% and 96%, respectively. In comparison, the sensitivity and specificity of the MSI Analysis System were 67% and 100%; and for the NCI panel, 75% and 97%. The difference in sensitivity between the LMR panel and the other panels was statistically significant (p<0.001). The increased sensitivity for detection of MSI-High phenotype in early colorectal lesions with the new LMR markers indicates that MSI screening for the early detection of Lynch syndrome might be feasible.

Capillary electrophoresis (CE) and multiplex amplification with fluorescent tagging have been routinely used for STR typing in forensic genetics. However, CE-based methods restrict the number of markers that can be multiplexed simultaneously and cannot detect any intra-repeat variations within STRs. Several studies already have indicated that massively parallel sequencing (MPS) may be another potential technology for STR typing. In this study, the prototype PowerSeq™ Auto System (Promega) containing the 23 STR loci and amelogenin was evaluated using Illumina MiSeq. Results showed that single source complete profiles could be obtained using as little as 62pg of input DNA. The reproducibility study showed that the profiles generated were consistent among multiple typing experiments for a given individual. The mixture study indicated that partial STR profiles of the minor contributor could be detected up to 19:1 mixture. The mock forensic casework study showed that full or partial profiles could be obtained from different types of single source and mixture samples. These studies indicate that the PowerSeq Auto System and the Illumina MiSeq can generate concordant results with current CE-based methods. In addition, MPS-based systems can facilitate mixture deconvolution with the detection of intra-repeat variations within length-based STR alleles.

STR typing in forensic genetics has been performed traditionally using capillary electrophoresis (CE). However, CE-based method has some limitations: a small number of STR loci can be used; stutter products, dye artifacts and low level alleles. Massively parallel sequencing (MPS) has been considered a viable technology in recent years allowing high-throughput coverage at a relatively affordable price. Some of the CE-based limitations may be overcome with the application of MPS. In this study, a prototype multiplex STR System (Promega) was amplified and prepared using the TruSeq DNA LT Sample Preparation Kit (Illumina) in 24 samples. Results showed that the MinElute PCR Purification Kit (Qiagen) was a better size selection method compared with recommended diluted bead mixtures. The library input sensitivity study showed that a wide range of amplicon product (6-200ng) could be used for library preparation without apparent differences in the STR profile. PCR sensitivity study indicated that 62pg may be minimum input amount for generating complete profiles. Reliability study results on 24 different individuals showed that high depth of coverage (DoC) and balanced heterozygote allele coverage ratios (ACRs) could be obtained with 250pg of input DNA, and 62pg could generate complete or nearly complete profiles. These studies indicate that this STR multiplex system and the Illumina MiSeq can generate reliable STR profiles at a sensitivity level that competes with current widely used CE-based method.

Quantitative real-time PCR (qPCR) assays have been widely adopted for molecular diagnostics over the past 15 to 20 years. These assays are used to evaluate gene expression, to detect and quantitate infectious agents, and for genotyping. Probe-based methods are more commonly used than double stranded DNA binding dye-based methods in diagnostic assays because of their improved specificity. Both amplification primers and the probe must anneal to the region of interest to generate a fluorescent signal, providing a third level of specificity not achieved with dye-based methods. Although dye-based methods can be used for diagnostic assays, extensive effort should be undertaken to ensure the primers are specific to the target of interest and do not form primer-dimers. The most widely used probe-based assays use hydrolysis probes (eg, TaqMan assays; ABI–Life Technologies, Grand Island, NY) which were first described in 1991.1Holland P.M. Abramson R.D. Watson R. Gelfand D.H. Detection of specific polymerase chain reaction product by utilizing the 5′→3′ exonuclease activity of Thermus aquaticus DNA polymerase.Proc Natl Acad Sci U S A. 1991; 88: 7276-7280Crossref PubMed Scopus (2177) Google Scholar The method began to gain traction in the mid-1990s with the development of quenched, fluorescent probes2Lee L.G. Connell C.R. Bloch W. Allelic discrimination by nick-translation PCR with fluorogenic probes.Nucleic Acids Res. 1993; 21: 3761-3766Crossref PubMed Scopus (632) Google Scholar, 3Livak K.J. Flood S.J. Marmaro J. Giusti W. Deetz K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. 1995; 4: 357-362Crossref PubMed Scopus (1330) Google Scholar and the commercialization of real-time thermal cyclers.4Heid C.A. Stevens J. Livak K.J. Williams P.M. Real time quantitative PCR.Genome Res. 1996; 6: 986-994Crossref PubMed Scopus (5010) Google Scholar, 5Gibson U.E. Heid C.A. Williams P.M. A novel method for real time quantitative RT-PCR.Genome Res. 1996; 6: 995-1001Crossref PubMed Scopus (1777) Google Scholar Alternative probe-based technologies employ molecular beacons,6Tyagi S. Kramer F.R. Molecular beacons: probes that fluoresce upon hybridization.Nat Biotechnol. 1996; 14: 303-308Crossref PubMed Scopus (3641) Google Scholar Scorpions probes,7Whitcombe D. Theaker J. Guy S.P. Brown T. Little S. Detection of PCR products using self-probing amplicons and fluorescence.Nat Biotechnol. 1999; 17: 804-807Crossref PubMed Scopus (621) Google Scholar HyBeacons probes,8French D.J. Archard C.L. Brown T. McDowell D.G. HyBeacon probes: a new tool for DNA sequence detection and allele discrimination.Mol Cell Probes. 2001; 15: 363-374Crossref PubMed Scopus (83) Google Scholar or other chemistries for simultaneous fluorescent detection of accumulating amplification products. Although hydrolysis probes and molecular beacons work well in properly designed assays, it can be a challenge to develop robust assays. The challenges are evidenced by the fact that Applied Biosystems undertook the effort to inventory more than 1.3 million monoplex TaqMan assays. The difficulty is compounded when attempting to design multiplexed assays. Furthermore the costs associated with dual-labeled probes can be prohibitively expensive when designing a new assay, especially in instances when several different probes must be synthesized and tested to derive the most robust assay. In this issue of The Journal of Molecular Diagnostics, Murray et al9Murray J.L. Hu P. Shafer D.A. Seven novel probe systems for real time PCR providing absolute single base discrimination, higher signaling, and generic components.J Mol Diagn. 2014; 16: 627-638Scopus (11) Google Scholar describe seven new probe-based approaches for qPCR that overcome some of the complications and expense associated with designing new qPCR assays. The approaches fall into two categories: internal probes (internal DNA Detection Switch, MacMan and Flip probes) and primer-probes (ZIPR, Universal, Half-Universal, and G-Force probes). This new suite of detection technologies (GeneTAG Technology, Inc., Atlanta, GA) offers comparable or improved performance relative to commonly used qPCR detection methods, with the potential to reduce oligonucleotide synthesis costs, simplify assay design, and improve assay specificity. The internal DNA Detection Switch employs a fluorescently-labeled probe that is complementary to the target and a quencher-labeled antiprobe containing a mismatch. The mismatched nucleotide allows for preferential binding of the probe to the desired target DNA, but reduces the incidence of hybridization to mismatched sequences (eg, regions containing single nucleotide polymorphisms). This configuration eliminates the requirement for a dual labeled probe and increases specificity for the target. Murray et al9 provide experimental evidence demonstrating the ability to reliably distinguish single nucleotide polymorphisms. The internal DNA Detection Switch maintains specificity across a 23°C temperature range. In contrast, a TaqMan probe targeting the same polymorphism loses specificity on a 3°C change in annealing temperature. Because the internal DNA Detection Switch can tolerate a broad range of annealing temperatures while maintaining specificity, Murray et al9Murray J.L. Hu P. Shafer D.A. Seven novel probe systems for real time PCR providing absolute single base discrimination, higher signaling, and generic components.J Mol Diagn. 2014; 16: 627-638Scopus (11) Google Scholar suggest the design of robust multiplexed assays is less dependent on optimally matching the thermal melt temperature of multiple probes. Matching thermal melt temperatures can be particularly challenging when targeting regions containing different GC% content. The second approach (MacMan) also uses a fluorescently-labeled probe and an antiprobe that is labeled with a quencher. The antiprobe anneals to the fluorescent probe via a region on the 3′ end of the probe that lacks homology to known sequences. When bound, the complex is nonfluorescent. On hybridization to the amplicon, the quencher and fluorescent label are spatially separated resulting in a fluorescent signal. The advantage of this approach is that one antiprobe construct can be combined with a number of sequence-specific probes, reducing costs associated with oligonucleotide synthesis and simplifying assay design. A Flip probe comprises a 3′ fluorescently-labeled probe and a 5′ quencher-labeled antiprobe joined to a sequence-specific probe via a glycol spacer. The antiprobe is complementary to the labeled probe, resulting in signal quenching in the absence of the desired amplification product. As the name implies, on annealing to the desired amplicon, the probe will flip-forward and emit a fluorescent signal. Annealing of the probe to the amplicon is the more thermodynamically stable conformation because the probe is several bases longer than the antiprobe. This design alters the amplification kinetics, yielding a linear signal increase on product accumulation. Because product accumulation results in a linear increase in the fluorescent signal, as opposed to the plateau normally associated with a TaqMan assay, the approach is ideally suited for end-point assays. The ZIPR primer-probe system takes advantage of the increased specificity inherent with the internal DNA Detection Switch system. With the ZIPR primer-probe approach, the target-specific primer contains a fluorescent label at the 5′ end. The separate antiprobe contains a 3′ quencher, is partially complementary to the labeled primer, but contains an intentional mismatch. Because of the mismatch, primer annealing to a perfectly complementary target (and subsequent extension by the polymerase) is the favored reaction. Mispriming events are minimized because the antiprobe anneals to the primer in the absence of a perfectly complementary target. By combining the ZIPR primer-probe and internal DNA Detection Switch system, Murray et al9Murray J.L. Hu P. Shafer D.A. Seven novel probe systems for real time PCR providing absolute single base discrimination, higher signaling, and generic components.J Mol Diagn. 2014; 16: 627-638Scopus (11) Google Scholar were able to interrogate two different locations in an amplicon. Targeting two different locations of the gene or infectious agent of interest allows for one region to serve as an internal control to confirm the presence and quantity of the target (ZIPR) and the second to interrogate a polymorphic region (internal DNA Detection Switch). Universal primer probe assays employ four different primers/probes: i) a target-specific, unlabeled primer that serves as one of the amplification primers; ii) a second target-specific, unlabeled amplification primer that includes a 5′ generic tail (linker-primer); iii) a fluorescently-labeled universal primer that is homologous to the generic tail of the second target-specific amplification primer (universal primer); and iv) a quencher-labeled antiprobe that is complementary to the universal primer. Inclusion of a quencher-labeled antiprobe ensures fluorescence will be observed only when the universal primer is extended to generate the desired amplicon. The concentration of the universal primer is 10-fold greater than the concentration of the linker primer. Consequently, the unlabeled primers participate in early cycles of amplification, but the universal primer is favored over the linker-primer in subsequent amplification cycles. This approach has the advantage of allowing a single labeled universal primer/antiprobe pair to be used for multiple assays, significantly reducing oligonucleotide synthesis costs. The half-universal probe is comparable to the linker-primer used in the Universal primer probe assays, but includes a 5′ fluorescent label. The quencher-labeled antiprobe ensures that only amplification products emit a fluorescent signal (ie, no fluorescence from unextended primer). Because all of the amplicons are directly labeled, the authors observe greater sensitivity relative to established TaqMan assays. Finally, the G-Force probe system is based on a self-quenching primer. The 5′ fluorescently-labeled primer contains cytosine-rich and guanine-rich segments that are separated by a hexa-ethyleneglycol spacer. The oligonucleotide assumes secondary structure due to hydrogen-bonding between the cytosine-rich and guanine-rich segments. The adopted structure brings the fluorescent label into close proximity with the guanine-rich segment and the fluorescent signal is quenched by photo-induced electron transfer.10Crockett A.O. Wittwer C.T. Fluorescein-labeled oligonucleotides for real-time PCR: using the inherent quenching of deoxyguanosine nucleotides.Anal Biochem. 2001; 290: 89-97Crossref PubMed Scopus (198) Google Scholar, 11Doose S. Neuweiler H. Sauer M. Fluorescence quenching by photoinduced electron transfer: a reporter for conformational dynamics of macromolecules.Chemphyschem. 2009; 10: 1389-1398Crossref PubMed Scopus (376) Google Scholar, 12Noble J.E. Wang L. Cole K.D. Gaigalas A.K. The effect of overhanging nucleotides on fluorescent properties of hybridizing oligonucleotides labelled with Alexa-488 and FAM fluorophores.Biophys Chem. 2005; 113: 255-263Crossref PubMed Scopus (35) Google Scholar During amplification, the primer is incorporated into a double-stranded amplicon, eliminating the secondary structure and allowing fluorescence emission. Murray et al9Murray J.L. Hu P. Shafer D.A. Seven novel probe systems for real time PCR providing absolute single base discrimination, higher signaling, and generic components.J Mol Diagn. 2014; 16: 627-638Scopus (11) Google Scholar suggest that the G-Force probe system can be used in combination with the internal DNA Detection Switch system, comparable to using a combination of ZIPR and internal DNA Detection Switch primer-probe systems to interrogate two different regions of the target of interest. Both approaches have the distinct advantage of using one region of the target of interest as a means to confirm the presence and quantity of the target, while the other region is used to interrogate potential polymorphisms. Although qPCR assays have been broadly adopted for gene expression analysis, pathogen detection and quantitation, and genotyping, most of the approaches have relied on a limited set of tools (primarily hydrolysis probes and molecular beacons). Murray et al9Murray J.L. Hu P. Shafer D.A. Seven novel probe systems for real time PCR providing absolute single base discrimination, higher signaling, and generic components.J Mol Diagn. 2014; 16: 627-638Scopus (11) Google Scholar describe an entirely new tool set that offers comparable or improved performance relative to the predominate approaches for qPCR. This new tool set should reduce the cost of designing robust assays, enable improved multiplexing, and allow for increased stringency in genotyping assays. The internal probe systems, especially the internal DNA Detection Switch, potentially represent a significant improvement over hydrolysis probe methods for genotyping assays. The primer-probe systems have a potential for false priming, as with other assays that rely on two levels of specificity (eg, dye-based qPCR). The inclusion of antiprobes minimizes the potential for false priming. Furthermore, when the primer-probes are coupled with the internal probe systems, the impact of false priming is significantly reduced. Although there is limited demonstration of clinical use, Murray et al9Murray J.L. Hu P. Shafer D.A. Seven novel probe systems for real time PCR providing absolute single base discrimination, higher signaling, and generic components.J Mol Diagn. 2014; 16: 627-638Scopus (11) Google Scholar note they plan to publish clinical validation studies in the near future.