Figure 5 - uploaded by Mogens Havsteen Jakobsen
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An illustration of the microtiter plate for the apoE genotyping assay on patient samples. Codon 112 CGC (purple strip), codon 112 TGC (clear strip), codon 158 CGC (green strip) and codon 158 TGC (yellow strip). The SNP position is underlined.
Source publication
Genotyping of single nucleotide polymorphisms (SNPs) in large populations presents a great challenge, especially if the SNPs
are embedded in GC‐rich regions, such as the codon 112 SNP in the human apolipoprotein E (apoE). In the present study, we have used immobilized locked nucleic acid (LNA) capture probes combined with LNA‐enhancer oligonucleoti...
Context in source publication
Context 1
... panel of patient samples previously genotyped for the apoE polymorphisms (24) (Fig. 4). Puri®ed genomic DNA from the patients was used as template in the ampli®cation of the apoE PCR products. The apoE genotyping assay resulted in clear, unambiguous results for all patient samples, con- curring with the genotypes obtained by DNA sequencing (Fig. 5). Table 3. The S/N ratio for LNA-enhancer entry 10 and the sequence identical DNA enhancer at various concentrations Table 4. Design of improved LNA-enhancer oligonucleotides e100 Nucleic Acids Research, 2002, Vol. 30 No. 19 ...
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Citations
... Thus, there is a great need to simultaneously detect those therapeutically actionable or driver mutations by ctDNA liquid biopsy in both circumstances when tissue specimens cannot be obtained repeatedly (15). To accurately detect low-level ctDNA and MRD, we thoroughly investigated available ctDNA detection technologies, such as widely used Amplification Refractory Mutation System (ARMS), digital PCR (dPCR)/digital droplet PCR (ddPCR) (16,17), Next-generation sequencing (NGS) technology (18,19), and several blocker sequence-based PCR enrichment methods (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). ARMS-PCR is clinically affordable, but it can only detect one mutation per assay and has a limit of detection ranging between ∼0.1 and 1% using 4 ml plasma (39,40). ...
... Although optimized to achieve high sensitivity, these NGS technologies require high sequencing depth (>10,000X) and is costly during periodical MRD testing. In consideration of both detection limits and accumulated MRD testing costs, we reviewed other simple, sensitive, and cost-effective technologies, which include peptide nucleic acid (PNA)-based method (23,29,34,36), locked nucleic acid (LNA)-based method (21,22,25,26,28,31,32), dual priming oligonucleotide (DPO)-based method (20,24,27,30,33,35,37), and blocker displacement amplification (BDA) (38). Except for BDA, the mentioned approaches used modified blocker sequence with outperformed affinity to bind with the complementary sequence other than the sequence with a certain mutation. ...
Circulating tumor DNA (ctDNA), a tumor-derived fraction of cell-free DNA (cfDNA), has emerged as a promising marker in targeted therapy, immunotherapy, and minimal residual disease (MRD) monitoring in postsurgical patients. However, ctDNA level in early-stage cancers and postsurgical patients is very low, which posed many technical challenges to improve the detection rate and sensitivity, especially in the clinical practice of MRD detection. These challenges usually include insufficient DNA input amount, limit of detection (LOD), and high experimental costs. To resolve these challenges, we developed an ultrasensitive ctDNA MRD detection system in this study, namely PErsonalized Analysis of Cancer (PEAC), to simultaneously detect up to 37 mutations, which account for 70–80% non-small cell lung cancer (NSCLC) driver mutations from low plasma sample volume and enables LOD of 0.01% at a single-site level. We demonstrated the high performance achieved by PEAC on both cfDNA reference standards and clinical plasma samples from three NSCLC patient cohorts. For cfDNA reference standards, PEAC achieved a specificity of 99% and a sensitivity of 87% for the mutations at 0.01% allele fraction. In the second cohort, PEAC showed 100% concordance rate between ddPCR and Next-generation sequencing (NGS) among 29 samples. In the third cohort, 22 of 59 patients received EGFR TKI treatment. Among them, three in four patients identified low level actionable gene mutations only by PEAC had partial responses after targeted therapy, demonstrating high ctDNA detection ability of PEAC. Overall, the developed PEAC system can detect the majority of NSCLC driver mutations using 8–10 ml plasma samples, and has the advantages of high detection sensitivity and lower costs compared with the existing technologies such as ddPCR and NGS. These advantages make the PEAC system quite appropriate for ctDNA and MRD detection in early-stage NSCLC and postsurgical recurrence monitoring.
... To generate the fluorescence signal, sequence non-specific intercalating dyes or sequence-specific probes such as hydrolysis probes, locked nucleic acid probes or molecular beacons 27,84,[87][88][89] can be used. The locked nucleic acid probes base on standard hydrolysis probes but have a higher melting temperature resulting in a higher sequences specificity 90,91 . The latter is important to be able to detect small changes in the DNA sequence as it is for the single nucleotide polymorphisms (SNPs) or point mutations. ...
Novel, easily accessible biomarkers in body fluids with the so-called liquid biopsy have come into focus in cancer research. This is especially the case for circulating tumor DNA in patients’ blood streams. The simplicity of a blood sampling makes continuous monitoring of tumor status possible. The surveillance of the patient’s mutation pattern during treatment is essential to select the most effective therapy. This work aims to demonstrate the use of centrifugal microfluidics for colorectal cancer monitoring by liquid biopsy. For this, the complete workflow from circulating cell-free DNA (cfDNA) extraction to the quantification of point mutations by multiplexed digital droplet PCR (ddPCR) is demonstrated.
Today’s standard methods for cfDNA extraction in a clinical environment are automated pipetting robots. However, the high purchasing costs, the precious laboratory space and lack of experts are a major problem, especially in smaller hospitals. This work shows a concept for an automated cfDNA extraction workflow in a monolithically integrated cartridge with pre-stored reagents in a small benchtop device. For the integration of the current gold standard assays for cfDNA extraction, it had to be transferred from a spin-column based workflow to a bead-based approach. Here, the most crucial influencing factors such as bead type and its volume, binding time, ethanol concentration during washing, drying after washing to remove remaining ethanol and cooling before DNA binding were all optimized in a way, that the bead-based approach yields the same results as the workflow with the spin-column. This means, that the bead-based approach in the manual workflow shows extraction recoveries close to the one from the spin-columns with approx. 100% recovery efficiency compared to the control.
Furthermore, the integration of the manual bead-based cfDNA extraction assay into the LabDisk was successfully shown. Independent of the DNA concentration (104-102 DNA copies/ml plasma) spiked with synthetic mutant DNA into the plasma sample the achievable recovery for the LabDisk had an average of 95% (range 83% - 108%). The LabDisk showed the advantage of the automation with a higher reproducibility in DNA recovery rates shown in a lower standard deviation (±13%) compared to the manual approaches with ±17% (spin-column) and ±26% (beads). Comparison of automated cfDNA extraction on the LabDisk with the QIAsymphony shows very comparable results for the patient plasma samples while at the same time reducing technical complexity. Consequently it is shown that the LabDisk is a highly attractive alternative to huge automation robots such as the QIAsymphony or to a tedious manual workflow in clinical routine workflow for liquid biopsy.
cfDNA quantification in a liquid biopsy context is most commonly performed by assessing comprehensive target panels to monitor the tumor burden of a cancer patient. In this work, optimization-free multiplex assays for ddPCR, with the use of the mediator probe (MP) technology are established for the seven most frequent point mutations in colorectal carcinoma (KRAS and BRAF). The limit of detection for the developed 2-plex and 4-plex MP ddPCR was measured with spiking experiments of synthetic mutant DNA in human genomic DNA. During therapy monitoring, the allele frequency which is the parameter to be determined. The MP ddPCR assays showed a limit of detection of 0.004% to 0.38% for the multiplex MP ddPCR assay with a R² of 0.98%. The limit of blank was determined to be 0 DNA copies/ml plasma for the 2-plex MP ddPCR assays whereas it was increased for KRAS G13D to be 9.82 DNA copies/ml plasma and for BRAF V600E to be 16.29 DNA copies/ml plasma in the 4-plex MP ddPCR assay. However, by the usage of patient plasma eluates, both MP ddPCR assay concepts (2-plex and 4-plex) were able to assign 100% of the tested patient samples correct positive or negative. Furthermore, they showed comparable performance to the reference locked nucleic acid assay from the university hospital Freiburg. The presented MP ddPCR assay needs no time consuming assay optimization as all presented assays uses the same concentrations for the PCR reagents and the same cycling conditions independent of the target to be analyzed.
However, a high degree of multiplexing is essential in cancer diagnostics to monitor for example clonal evolution during treatment. Therefore, this work shows an increased degree of multiplexing by the usage of virtual fluorescence color channels by introducing a photobleaching-resistant and photobleaching-sensitive assay concept for the quantification of point mutations. This combination leads to the doubling of the multiplexing degree for each channel. The photobleaching effect, requires an additional readout step and a low-cost light-emitting diode (LED). This concept can increase the degree of multiplexing of the used ddPCR reader from three standard channels to six channels by the introduction of three additional virtual channels by photobleaching of the fluorophores. The usability of this concept for liquid biopsy was demonstrated using the two cancer-associated point mutations KRAS G12D and G12V. With a serial dilution experiment in the red channel, the required sensitivity of assays used for liquid biopsy the limit of detection for KRAS G12D was determined to 16 DNA copies/reaction (standard channel) and for KRAS G12V to nine DNA copies/reaction (virtual channel).
As a final topic, the developed 4-plex MP ddPCR assay is integrated into a centrifugal microfluidic ddPCR cartridge. The latter is able to generate droplets with a diameter of approx. 82.7 µm in each of the 12 identical ddPCR unit. The oil-surfactant concentration and the oil-combination were determined to be the most crucial influencing parameter for a robust droplet generation and thermostable droplets during PCR. This ddPCR disk featured the usage of a standard fluorescence microscope for the evaluation of the fluorescence data from the ddPCR units. The integration of one of three 4-plex panels was demonstrated with KRAS wild type and the cancer-associated point mutations KRAS G12A, G12D and G12V. The limit of detection experiment showed the diagnostically relevant sensitivity with 3.5 mutant DNA copies in a background of 15,000 DNA copies from the wild type with a R² of 0.99.
With both the aforementioned cfDNA extraction the ddPCR assay for cfDNA quantification integrated into the LabDisk in this work, the entire liquid biopsy workflow could be automated for the first time using centrifugal microfluidics. Comparative experiments showed excellent performance of the disks and the assays compared to gold standard methods demonstrating the feasibility and great potential of this technology for future introduction into routine clinical practice.
... E484K and N501Y mutations were detected in the Cal Fluor Red 610 (Probe 484K) and Quasar 670 (Probe 501Y) channels, respectively. As the probes differ only on a single nucleotide, there will be hybridization competition during the alignment and extension phase of thermocycling, favoring the specific binding of probes that do not have a mismatch with their target (Jacobsen et al., 2002). For the N501Y mutation, modified bases (LNA) were used to allow better discrimination due to the low DTm of hybridization between the complementary base and the mismatch. ...
Background
Several variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population.
Methods
Targeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico.
Results
In silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico.
Conclusion
This work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.
... These LNAzymes are able to cleave the phosphodiester bond with improved ef iciency compared to DNAzymes. 19 Furthermore, LNA modiied oligonucleotides have been used as probes in luorescent in situ hybridization assays 20 , in microarray applications for single nucleotide polymorphism (SNP) detection 21,22 and as antisense oligonucleotides. 23 Phosphorothioates are chiral oligonucleotide mimics obtained by replacing a nucleotide's non-bridging phosphate oxygen by a sulphur atom. ...
... Daniel et al. [11] reported that mutant genotype of a C allele SNP of individuals predicted lower RBC folate concentration than that of T allele SNP of individuals with the same diet folate intake level. Studies have reported numbers of micronutrient deficiencyrelated SNPs (MD-SNPs) of vitamins A, D, E, B 6 , and B 12 , folate, calcium, iron, zinc, selenium, etc. [14][15][16][17][18][19][20][21][22]. It is assumed that MD-SNPs based on high -quality observations in large population could be used as biomarkers for assessing genetic potential risk of micronutrient deficiency. ...
Malnutrition is a result of complicated reasons from diet and food behavior and also related to genetic background which has been revealed by studies in recent decades. Traditionally, nutrition status are measured and expressed with indexes of anthropometric, diet survey, clinical symptom, biochemistry, behavior, etc. These measurement has been used in national nutrition monitoring, clinic nutrition therapy, mother and children nutrition care, nutrition intervention projects, and scientific studies. However, genetic and epigenetic information on nutrition explain malnutrition in a genetic view that would supply additional new theory and methodology for the growing requirement in terms of personalized and precise nutrition. In this chapter, an introduction on the detection of nutrient-related SNP to reveal individual malnutrition risk is discussed.
... We designed nine gapmer AONs targeting the c.166G>A mutation but shifted the oligonucleotides each time a few bps, as it is known that only a few bps difference can lead to a complete abolishment of inhibitory activity of AONs due to secondary or tertiary structures that restrict binding possibilities of the AON [32]. Furthermore, LNA modifications increase the difference in melting temperature between duplexes formed with a perfectly matched target versus a mismatched target and are thus possibly increasing the allelic-discrimination potency of AONs [33,34]. Hence, we included nine gapmer AONs containing a LNA modification at the site of the mutation. ...
The recurrent missense variant in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3), c.166G>A, p.(Gly56Arg) or G56R, underlies 1%–2% of cases with autosomal dominant retinitis pigmentosa (adRP), a frequent, genetically heterogeneous inherited retinal disease (IRD). The mutant NR2E3 protein has a presumed dominant negative effect (DNE) by competition for dimer formation with Cone-Rod Homeobox (CRX) but with abolishment of DNA binding, acting as a repressor in trans. Both the frequency and DNE of G56R make it an interesting target for allele-specific knock-down of the mutant allele using antisense oligonucleotides (AONs), an emerging therapeutic strategy for IRD. Here, we designed gapmer AONs with or without a locked nucleic acid modification at the site of the mutation, which were analyzed for potential off-target effects. Next, we overexpressed wild type (WT) or mutant NR2E3 in RPE-1 cells, followed by AON treatment. Transcript and protein levels of WT and mutant NR2E3 were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot respectively. All AONs showed a general knock-down of mutant and WT NR2E3 on RNA and protein level, showing the accessibility of the region for AON-induced knockdown. Further modifications are needed however to increase allele-specificity. In conclusion, we propose the first proof-of-concept for AON-mediated silencing of a single nucleotide variation with a dominant negative effect as a therapeutic approach for NR2E3-associated adRP.
... 70 The high specificity of LNA and high thermal stability in complex with DNA makes LNA-based probes well adapted for detection of nucleic acids. 71,72 In addition, because the physical properties of LNA are highly similar to that of DNA, it is possible to adapt LNA for use in experimental conditions optimized for DNA-based probes. Moreover, LNA can be readily commercially synthesized, either purely as LNA or with the addition of DNA or RNA bases. ...
Fluorescence in situ hybridization coupled with flow cytometry (FISH-Flow) is a highly quantitative, high-throughput platform allowing precise quantification of total mRNA transcripts in single cells. In undiagnosed infections posing a significant health burden worldwide, such as latent tuberculosis or asymptomatic recurrent malaria, an important challenge is to develop accurate diagnostic tools. Antigen-specific T cells create a persistent memory to pathogens, making them useful for diagnosis of infection. Stimulation of memory response initiates T-cell transitions between functional states. Numerous studies have shown that changes in protein levels lag real-time T-cell transitions. However, analysis at the single-cell transcriptional level can determine the differences. FISH-Flow is a powerful tool with which to study the functional states of T-cell subsets and to identify the gene expression profiles of antigen-specific T cells during disease progression. Advances in instrumentation, fluorophores, and FISH methodologies will broaden and deepen the use of FISH-Flow, changing the immunological field by allowing determination of functional immune signatures at the mRNA level and the development of new diagnostic tools.
... During PCR, LNA sequence selectively blocks amplification of wildtype DNA, while allowing the amplification of the mutant codon 48 . Currently, several LNA genotyping assays have been reported for the screening of i) factor V Leiden mutation, ii) apolipoprotein B (apoB) R3500Q mutation and iii) two mutations in apolipoprotein E [49][50][51] . In these assays, 8mer LNAcapture probes (complementary to either the wild type or the mutated genomic sequence) are covalently attached to individual wells of a microtiter plate and, after hybridization with PCR amplicons, scored colorimetrically with an ELISA-like technique 49 . ...
A AB BS ST TR RA AC CT T: : Background: Genotyping is crucial to the identification of genetic markers underlying development of neoplastic diseases and individual variations in responses to specific drugs. Cost-and time-effective technologies able to accurately identify genetic polymorphisms will dramatically affect routine diagnostics processes and future therapeutic developments. However, such methods need to fulfill the principles of analytical validation to determine their suitability to assess nucleotide polymorphisms in target genes. Approach: This article reviews the recent developments of technologies for genotyping of single nucleotide polymorphisms (SNPs). For the appropriate choice of any method, several criteria must be considered: i) known or unknown genetic variations in a given cancer gene; ii) needs of testing within pharmacogenomics studies; iii) diffusion and availability of large platforms and required equipments; iv) suitability of tests for routine diagnostics; v) capacity of methods to offer a specific and sensitive detection of mutant alleles within great excess of wild-type alleles in a given sample; vi) suitability for high-throughput implementation. Content: This review is intended to provide the reader with a better understanding of the various technologies for pharmacogenomics testing in the routine clinical laboratory. A brief overview is given on the available technologies for detection of known mutations together with a precise description of the homogeneous technologies and platforms currently employed in genotyping analysis. Based on the criteria proposed here, potential users may evaluate advantage and limitations of the different analytical platforms and possibly identify the most appropriate one according to specific operative settings and diagnostic needs.
... During PCR, LNA sequence selectively blocks amplification of wildtype DNA, while allowing the amplification of the mutant codon 48 . Currently, several LNA genotyping assays have been reported for the screening of i) factor V Leiden mutation, ii) apolipoprotein B (apoB) R3500Q mutation and iii) two mutations in apolipoprotein E [49][50][51] . In these assays, 8mer LNAcapture probes (complementary to either the wild type or the mutated genomic sequence) are covalently attached to individual wells of a microtiter plate and, after hybridization with PCR amplicons, scored colorimetrically with an ELISA-like technique 49 . ...
... 3,4 The latter approach can increase the difference in MT and WT duplex stabilities through the ability of LNA substitutions to decrease probe length while maintaining T m,MT , 4,5 and the generally greater energetic intolerance of LNAs to participate favorably in mismatched base pairs. 6 LNA-bearing probes have therefore been shown to offer specific advantages when applied to the detection of either germline variants, including singlenucleotide polymorphisms (SNPs), 4,7,8 or acquired variants, including somatic point mutations (SPMs). 9−11 MGB-and LNA-modified probes can demonstrate comparable performance in qPCR genotyping assays. ...
Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of Tm values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the Tm of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a quantitative PCR or dPCR assay. This potential is demonstrated by using the model to design allele-specific probes that completely discriminate and quantify clinically relevant mutant alleles (BRAF V600E and KIT D816V) in a dPCR assay.
