FAM-labeled Probe Research Articles

Fluorometric determination of RNase H via a DNAzyme conjugated to reduced graphene oxide, and its application to screening forinhibitors and activators.

A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg2+. The recycle cleavage can directly result in the amplifiedsignal emitted bytheFAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5U·mL-1. The detection limit of 0.018U·mL-1 is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screenedfor their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection. Graphical abstract The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.

Fluorometric determination of HIV DNA using molybdenum disulfide nanosheets and exonuclease III-assisted amplification.

A convenient and ultrasensitive fluorometric method is described for the determination of HIV DNA. It exploits the strong difference in the affinities of MoS2 nanosheets for long ssDNA versus short oligonucleotide fragments. In addition, efficient signal amplification is accomplished by exonuclease III-assisted target recycling. When absorbed on the MoS2 nanosheets, the fluorescenceof the FAM-labeled ssDNA probe (FP) is quenched. However, in the presence of HIV DNA, the FP hybridizes with target to form a duplex. As a result, the FP in the duplex will be stepwise hydrolyzed into short fragments by Exo III, and the fluorescence signal thus isretained because short fragments have low affinity for the MoS2 nanosheets. By using the Exo III-assisted target recycling amplification, the detection sensitivity is strongly improved. The sensor can detect DNAin a concentration as low as 5.3 pM (at an S/N ratio of 3), and the analytical range extends from 0.01nM to 10nM. The assay is simple, sensitive and specific, and conceivably represents a valuable tool in clinical studies related to the HIV. Graphical abstract Schematic presentation of fluorometric determination of HIV DNA based on molybdenum disulfide nanosheets and Exo III. When the fluorescence-tagged ssDNA probe hybridized with target to form a duplex, the Exo III-assisted target recycling amplification is generated. The method can detect as low as 5.3 pM HIV DNA.

A dynamic, ultra-sensitive and “turn-on” strategy for fluorescent detection of uranyl based on DNAzyme and entropy-driven amplification initiated circular cleavage amplification

A uranyl detection strategy with ultra-sensitivity was developed based on entropy-driven amplification and DNAzyme circular cleavage amplification. The cleavage of UO22+-specific DNAzyme produces a DNA fragment to initiate the entropy-driven amplification. Two DNA sequences released from the entropy-driven amplification are partly complementary. They can form an entire enzyme strand (E-DNA) of Mg2+-specific DNAzyme. The formed E-DNA can circularly cleave FAM-labeled probes on gold nanoparticles (AuNPs), causing the leaving of FAM from AuNPs and recovery of fluorescent signal. A linear relationship was obtained in the range from 30 pM to 5 nM between fluorescence intensity and concentration of UO22+. The limit of detection was low to 13 pM. This method showed a promising future for practical application in real water samples.

Abstract 16753: Efficacy & Efficiency of Genome Editing With CRISPR/Cas9 in a Humanized Knock-In Mouse Model of PLN R14del

Introduction: A mutation in the Phospholamban (PLN) gene, a calcium regulating protein, the Arg14 deletion (R14del), has been identified in patients with atypical Arrhythmogenic Cardiomyopathy (ACM). Carriers of this mutation are at high risk for malignant ventricular arrhythmias and ventricular dilation. Objective: To evaluate the efficiency and efficacy of genome editing by using CRISPR/Cas9 and a cardiotropic Adeno-Associated Vector (AAV) in a knock-in mouse model expressing the human coding sequence of mutant PLN (hPLN R14del ). Methods: We generated wild-type (WT), heterozygous (Het) and double mutant (Mut) humanized mice. We designed a CRISPR-Cas9/gRNA construct which cut around the site encoding R14Del-PLN gene. To check for CRISPR-Cas9 efficiency we used droplet digital (ddPCR) Genome Edit Detection assay that consist of primer pair to amplify the sequence that includes the mutation, and a FAM-labeled probe designed to bind the mutant allele. Heterozygous mice were injected with 2x10 12 vg of AAV9.CRISPR/Cas9/gRNA for inactivation of the R14del allele. Cardiac function and heart morphology were assessed by Magnetic Resonance Imaging (MRI), echocardiography and H&E staining. Ventricular tachycardia (VT) threshold was defined as the pacing frequency (f Pacing ) required for induction of sustained VT in ex vivo set-up. Results: Echocardiography and histology showed no significant changes in cardiac function and structure in the R14del Het & Mut mice. Eight weeks after AAV injection the hearts were harvested and analyzed for indel frequencies at on-target site in the genome. ddPCR analysis showed an efficiency of ~13% inactivation of the R14del mutation in AAV9/CRISPR-Cas9 injected mice compared to controls. Seventy-five percent of Het hearts exhibited adrenergic mediated VT (f Pacing = 19.6±4.7Hz) vs. only 30% of their WT counterparts (25.8±1.4Hz, p<0.05). VT susceptibility in Het hearts was significantly suppressed by AAV-mediated CRISPR/Cas9 delivery targeting the mutant allele. Conclusions: Our results demonstrate that mouse model harboring the human R14del mutation does not result in gross cardiac morphological changes. Silencing this mutation by CRISPR/Cas9 AAV based gene therapy reversed the VT susceptibility in the Het mice.

Abstract 4578: Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST)

Abstract Introduction: Genotype-directed therapies are revolutionizing care for gastrointestinal stromal tumors (GIST). Somatic mutations in KIT exons (ex) 11 and 9 induce constitutive kinase activity and account for ~95% of the primary oncogenic events in KIT-driven GIST; KIT mutations correlate with prognosis and predict clinical activity of tyrosine kinase inhibitors (TKI), such as imatinib. After a median of 24 months on imatinib, 85% of patients (pts) develop resistance and progress multifocally, mostly due to secondary KIT mutations in ex 13 and/or ex 17. We describe here a rapid, clinical grade droplet digital PCR (ddPCR) assay to detect and quantify KIT mutations associated with TKI resistance in cfDNA from plasma of metastatic (met) GIST pts. Unlike allele-specific ddPCR that requires splitting input DNA into many reactions, we designed pan-ex 17 KIT assay to capture the polyclonal mutational landscape of KIT in a single reaction. Methods: Tumor and plasma samples and clinical data were collected from pts with met GIST. ddPCR assays were developed for the detection in cfDNA of all common secondary KIT mutations associated with imatinib resistance (V654A, T670I, A829P and multiple ex 17 mutations). The pan-ex 17 mutation drop-off assay uses a FAM-labeled mutant (mut) probe that spans sequences encoding amino acids 819~824, and a VIC-labeled reference probe that is shared by both wild-type (wt) and mut alleles. Common ex 17 mutations were detected via cluster separation. V654A, T670I, A829P are detected with allele-specific assays that use a FAM-labeled mut probe and a VIC-labeled wt probe. To demonstrate analytical sensitivity/specificity of each assay, ddPCR cycling conditions were optimized to yield the maximum fluorescence signal with minimal noise signal. Results: The KIT ddPCR assays detect a mutation prevalence of 0.01 - 0.05%, with a sensitivity of 5 - 50 KIT mut copies in a background of 10,000 KIT wt copies, depending on the mutation assayed, with absolute specificity. Results were validated in a pilot observational cohort of pts who were treated with imatinib and progressed, for which matching plasma (1 ml) and tumor samples were analyzed to reveal identical mutations. In a separate cohort, ddPCR results were validated by sensitive plasma NGS. Conclusions: We have developed rapid, highly sensitive, and 100% specific quantitative assays to enable plasma-based tumor genotyping for secondary KIT resistance mutations, which has been technically optimized for translation into clinical practice. Analysis of cfDNA by ddPCR can successfully detect KIT secondary mutations in met GIST pts. In an ongoing study, serial monitoring of KIT mutations in cfDNA from pts with advanced GIST should allow for early detection of secondary mutations and optimization of genotype-targeted therapeutic management. Citation Format: Adrian Marino-Enriquez, Suzanne George, Julianna Supplee, Grace Heavey, Pasi A. Janne, Chandrajit Raut, Jonathan Fletcher, George D. Demetri, Cloud P. Paweletz, Yanan Kuang. Development of a rapid clinical grade assay to detect and monitor secondary KIT mutations in circulating free DNA (cfDNA) for personalization of targeted therapy for gastrointestinal stromal tumors (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4578.

Detection of Nucleic Acids in Complex Samples via Magnetic Microbead-Assisted Catalyzed Hairpin Assembly and "DD-A" FRET.

Nucleic acids, as one kind of significant biomarker, have attracted tremendous attention and exhibited immense values in fundamental studies and clinical applications. In this work, we developed a fluorescent assay for detecting nucleic acids in complex samples based on magnetic microbead (MMB)-assisted catalyzed hairpin assembly (CHA) and a donor donor-acceptor fluorescence resonance energy transfer ("DD-A" FRET) signaling mechanism. Three types of DNA hairpin probes were employed in this system, including Capture, H1 (double FAM-labeled probe as FRET donor), and H2 (TAMRA-labeled probe as FRET acceptor). First, the Captures immobilized on MMBs bound to targets in complex samples, and the sequences in Captures that could trigger catalyzed hairpin assembly (CHA) were exposed. Then, target-enriched MMB complexes were separated and resuspended in the reaction buffer containing H1 and H2. As a result, numerous H1-H2 duplexes were formed during the CHA process, inducing an obvious FRET signal. In contrast, CHA could not be triggered, and the FRET signal was weak, while target was absent. With the aid of magnetic separation and "DD-A" FRET, errors from background interference were effectively eliminated. Importantly, this strategy realized amplified detection in buffer, with detection limits of microRNA as low as 34 pM. Furthermore, this method was successfully applied to detect microRNA-21 in serum and cell culture media. The results showed that our method has the potential for biomedical research and clinical application.

A sensitive detection assay based on signal amplification technology for Alzheimer's disease's early biomarker in exosome

Alzheimer's disease (AD) considered as the third health “killer” has seriously threatened the health of the elderly. However, the modern diagnostic strategies of AD present several disadvantages: the low accuracy and specificity resulting in some false-negative diagnoses, and the poor sensitivity leading to a delayed treatment. In view of this situation, a enzyme-free and target-triggered signal amplification strategy, based on graphene oxide (GO) and entropy-driven strand displacement reaction (ESDR) principle, was proposed. In this strategy, when the hairpin structure probes (H)specially binds with beta-amyloid-(1–42) oligomers (Aβ42 oligomers), it's structure will be opened, causing the bases complementary to FAM-labeled replacement probes R (R1 and R2) exposed. At this time, R1 and R2 will hybridize with H, resulting in the bound Aβ42 oligomers released. The released Aβ42 oligomers would participate in the next cycle reaction, making the signal amplified. As a quencher, GO could absorb the free single-stranded DNA R1 and R2 and quench their fluorescence; however, the DNA duplex still exists free and keeps its signal-on. Through the detection of Aβ42 oligomers in exosomes, this ultrasensitive detection method with the advantages of low limit of detection (LOD, 20 pM), great accuracy, excellent precision and convenience provides an excellent prospect for AD's early diagnosis.

An “off-on” fluorescent switch assay for microRNA using nonenzymatic ligation-rolling circle amplification

The authors describe a fluorometric switch assay for microRNA (miRNA). It is based on nonenzymatic ligation-rolling circle amplification (NELRCA). A click chemistry-mediated functionalized linear template was prepared by self-cycling. It is capable of forming a modified circular template (MLT) with a triazole linkage for amplified agent. MiRNA can recognize a binding region of the MLT, activates the NELRCA, and converts into rolling circle amplification products (RCPs). Subsequently, the added FAM-labeled signal probes (FAM-SPs) can hybridize to the RCPs. This RCPs/FAM-SPs duplex is less adsorbed onto the graphene oxide (GO) so that fluorescence (best measured at excitation/emission wavelengths of 490/520 nm) increases. In contrast to classical GO-based adsorption and displacement sensing system, the designed hybridization and adsorption fluorescent switch-on platform can reduce the assay time, and improve the detection efficiency of target. Under optimal conditions, the sensing platform has a detection limit as low as 0.75 fM. It can well discriminate target miRNA from other kinds of miRNA. Conceivably, this method can be extended to the trace detection of other nucleic acid biomarkers, and simultaneous recognition of multiple targets, thereby improving the early clinical diagnostic accuracy of cancer.

Aptamer based fluorescent cocaine assay based on the use of graphene oxide and exonuclease III-assisted signal amplification

The article reports an aptamer based assay for cocaine by employing graphene oxide and exonuclease III-assisted signal amplification. It is based on the following scheme and experimental steps: (1) Exo III can digest dsDNA with blunt or recessed 3-terminus, but it has limited activity to ssDNA or dsDNA with protruding 3-terminus; (2) GO can absorb the FAM-labeled ssDNA probe and quench the fluorescence of probe, while the affinity between FAM-labeled mononucleotide and GO is negligible; (3) Cocaine aptamer can be split into two flexible ssDNA pieces (Probe 1 and Probe 2) without significant perturbation of cocaine-binding abilities; (4) The triple complex consisting of Probe 1, Probe 2 and cocaine can be digested by Exo III with the similar efficiency as normal dsDNA. Cocaine aptamer is split into two flexible ssDNA pieces (Probe 2 and 3′-FAM-labeled Probe 1). Cocaine can mediate the cocaine aptamer fragments forming a triplex. The triple complex has unique characteristic with 3′-FAM-labeled blunt end at the Probe 1 and 3′-overhang end at Probe 2. If exonuclease III is added, it will catalyze the stepwise removal of fluorescein (FAM) labeled mononucleotides from the 3-hydroxy termini of the special triplex complex, resulting in liberation of cocaine. The cocaine released in this step can produce a new cleavage cycle, thereby leading to target recycling. Through such a cyclic bound-hydrolysis process, small amounts of cocaine can induce the cleavage of a large number of FAM-labeled probe 1. The cleaved FAM-labeled mononucleotides are not adsorbed on the surface of graphene oxide (GO), so a strong fluorescence signal enhancement is observed as the cocaine triggers enzymatic digestion. Under optimized conditions, the assay allows cocaine to be detected in the 1 to 500 nM concentration range with a detection limit of 0.1 nM. The method was applied to the determination of cocaine in spiked human plasma, with recoveries ranging from 92.0 to 111.8 % and RSD of <12.8 %.

A novel diagnostic approach to detecting porcine epidemic diarrhea virus: The lateral immunochromatography assay

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in sucking piglets and has a high mortality rate. An immunochromatography (IC) assay, known as a lateral flow test, is a simple device intended to detect the presence of target pathogens. Here, we developed an IC assay that detected PEDV antigens with 96.0% (218/227) sensitivity and 98.5% (262/266) specificity when compared with real-time reverse transcriptase (RT)-PCR using FAM-labeled probes based on sequences from nucleocapsid genes. The detection limits of the real-time RT-PCR and IC assays were 1×102 and 1×103 copies, respectively. The IC assay developed herein did not detect non-specific reactions with other viral or bacterial pathogens, and the assay could be stored at 4°C or room temperature for 15 months without affecting its efficacy. Thus, the IC assay may result in improved PED detection and control on farms, and is a viable alternative to current diagnostic tools for PEDV.

IFNAR1 is a predictor for overall survival in colorectal cancer and its mRNA expression correlated with IRF7 but not TLR9.

Toll-like receptor (TLR) 9 plays a role in intestinal inflammation that, in turn, is related to the tumorigenesis of colorectal cancer. Nuclear factor κB (NFκB), and interferon regulatory factor (IRF) 5 and IRF7 can be activated by TLR9 and induce the production of proinflammatory cytokines and type I interferon, respectively. This study investigated the mRNA expressions of TLR9 and its downstream signaling molecules in both the tumor and the normal tissues of colorectal cancer.Eighty-four subjects with colorectal cancer were consecutively recruited at a community-based hospital, and the mRNA expression of TLR9, NFκB, IRF5, IRF7, interleukin 6 (IL6), and interferon α/β/ω receptor 1 (IFNAR1) in the tumor and normal tissue were determined by real-time reverse transcription polymerase chain reaction using TaqMan FAM-labeled MGB probes (Life Technologies, Carlsbad, CA).The tumor had higher percentages of detection of TLR9, IFNAR1, and IL6 mRNA expressions than normal tissue. The absence of detectable TLR9 mRNA expression was associated with an absence of significance in the correlation between IL6 and NFκB or IRF5, but not that between IRF7 and IFNAR1 in both the tumor and the normal tissues. An absence of detectable IFNAR1 mRNA expression in the tumor (hazard ratio: 3.77; 95% confidence interval: 1.22–11.60) and advanced stage (stages III and IV, 7.86; 1.76–35.40) were significant predictors for overall survival.IFNAR1 is a predictor for overall survival and mRNA expression is correlated to IRF7, but not TLR9 in colorectal cancer. The results cast doubt on the usefulness of TLR9 agonist in treating colorectal cancer.

HOXA11 and MMP2 gene expression in uterosacral ligaments of women with pelvic organ prolapse.

Pelvic organ prolapse (POP) is a common disorder that negatively impacts the quality of life in many women. Uterosacral ligaments (USLs) are supportive structures of the pelvic organs that are often attenuated in women with POP. The HOXA genes regulate the development of the uterosacral ligaments. We compared expression of HOXA11 and MMP2 in USLs of women with and without POP. A prospective sequential cross sectional study was conducted in ZTB Women's Health Research and Education Hospital. We compared expression of HOXA11 and MMP2 in USLs of women with (n:18) and without (n: 15) POP. Total RNA was isolated from patient (n:18) and control (n:15) uterosacral ligament tissues with TriPure isolation reagent according to the manufacturer's instructions. Expression levels of HOXA11 and MMP2 were determined using semiquantitative RT-PCR in a Light Cycler 480 system. Real-time ready catalog assays, which are short FAM-labeled hydrolysis probes containing locked nucleic acid, were used for RT-PCR reactions. There was no difference in patients' mean age, parity, body mass indexes, and menopausal status between two groups. Means of RNA expression of MMP2 were 1.27±0.6 and 0.75±0.4 in the POP group vs control group, respectively (p:0.007). Means of RNA expression of HOXA 11 were 2.57±2.4 and 1.94±1.4 in the POP group vs control group, respectively (p:0.376). The POP group was divided as mild and severe POP; there was no difference in HOXA11 and MMP2 RNA expression between groups (p>0.05). Although there was no difference HOXA11 RNA expression in USLs with the POP group vs control, there was a significant difference MMP2 RNA expression in USLs with the POP group vs control. There are limited studies on this subject, and study results are contradictory. Further investigations with larger numbers of cases are needed to clarify this subject.

Chemiluminescence resonance energy transfer biosensing platform for site-specific determination of DNA methylation and assay of DNA methyltransferase activity using exonuclease III-assisted target recycling amplification

Site-specific determination of DNA methylation and assay of MTase activity can be used for determining specific cancer types, providing insights into the mechanism of gene repression, and developing novel drugs to treat methylation-related diseases. Herein, we develop a simple and highly sensitive chemiluminescence (CL) biosensing platform for site-specific determination of DNA methylation using Exonuclease III (Exo III)-assisted target recycling signal amplification. After bisulfite treatment of mixture of methylated DNA and unmethylated DNA, methylated DNA can hybridize with fluorescein (FAM)-labeled probe DNA to form double-stranded DNA (dsDNA), removing the FAM-labeled probe DNA from the surface of grapheme oxide, and the chemiluminescence resonance energy transfer (CRET) sensing signal can be observed and then amplified using Exo III-based recycling strategy.The biosensing platform exhibits excellent high sensitivity, and it can ever distinguish as low as 0.002% methylation level from the mixture, which is superior to most currently reported methods used for DNA methylation assay. In addition, the proposed method can also be used to sensitively assay MTase activity with determination limit of 0.007U/mL. This CL biosensing offers the advantages of being facile, sensitive, rapid and cost-effective. These features make the system promising for future use for early cancer diagnosis and discover of new anticancer drugs.

An Improved SELEX-Seq Strategy for Characterizing DNA-Binding Specificity of Transcription Factor: NF-κB as an Example

SELEX-Seq is now the optimal high-throughput technique for characterizing DNA-binding specificities of transcription factors. In this study, we introduced an improved EMSA-based SELEX-Seq strategy with several advantages. The improvements of this strategy included: (1) using a FAM-labeled probe to track protein-DNA complex in polyacrylamide gel for rapidly recovering the protein-bound dsDNA without relying on traditional radioactive labeling or ethidium bromide staining; (2) monitoring the specificity of SELEX selection by detecting a positive and negative sequence doped into the input DNAs used in each round with PCR amplification; (3) using nested PCR to ensure the specificity of PCR amplification of the selected DNAs after each round; (4) using the nucleotides added at the 5′ end of the nested PCR primers as the split barcode to code DNAs from various rounds for multiplexing sequencing samples. The split barcode minimized selection times and thus greatly simplified the current SELEX-Seq procedure. The reliability of the strategy was demonstrated by performing a successful SELEX-Seq of a well-known transcription factor, NF-κB. Therefore, this study provided a useful SELEX-Seq strategy for characterizing DNA-binding specificities of transcription factors.

Real-time RT-PCR assay to differentiate clades of H5N1 avian influenza viruses circulating in Vietnam

Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 2.3.2.1, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.

Abstract 61: Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer.

Abstract Quantitative RT-PCR tests that measure transcript abundance of selected genes in clinical specimens promise to improve cancer diagnostic accuracy and enable “personalized medicine” through selection of the most effective treatment for each cancer. However, most clinical samples are Formalin-Fixed, Paraffin-Embedded (FFPE) and these yield sub-optimal RNA integrity. Thus, there is a need for qPCR tests with increased robustness that also have intrinsic quality control and low cost. To address this need, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of gene-specific internal standard (IS) molecules using two-color fluorometric analysis on real-time platform. For each gene, a competitive template IS was synthesized containing 4-6 nucleotide changes relative to the native template (NT). During PCR, NT or IS signal was quantified with sequence-specific FAM-labeled probe or Quasar 670-labeled probe, respectively. To optimize measurement sensitivity for analysis of highly degraded FFPE samples; a) each RNA sample was reverse transcribed (RT) with gene-specific primer and b) cDNA was pre-amplified with 18 PCR cycles in the presence of internal standards. Results obtained with pre-amp or no pre-amp conditions were highly correspondent. We conducted a validation study of this assay in 38 samples (10 malignant and 10 benign surgical FFPE tissues and 13 malignant and 5 benign fine needle aspirate (FNA) samples. Consistent with previous results in fresh frozen surgical samples, the lung cancer diagnostic test (LCDT) optimal cut-off value discriminated malignant from non-malignant tissues (p = 0.0009), had 92.9% specificity and 75.0% sensitivity, and a receiver operator characteristic area under the curve of 0.87 (95% confidence interval 0.74-0.99). Based on these data, we expect that this quality-controlled two color fluorometric qPCR approach will enable reliable analysis of the LCDT and other promising qPCR-based molecular diagnostic tests in small degraded RNA extracted from clinical FFPE and FNA cell block FFPE samples. Citation Format: Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, Lauren M. Stanoszek, Rachel E. Dannemiller, Laura Jordan, Jill Zyrek, Luis de la Casas, James C. Willey. Use of two-color fluorometric real-time PCR to develop molecular diagnostic tests with intrinsic quality control that augment cytomorphologic diagnosis of lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 61. doi:10.1158/1538-7445.AM2013-61

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.

Abstract 5541: Molecular diagnostic tests to augment cytomorphologic diagnosis of lung cancer

Abstract Morphologic analysis of cytology samples obtained by transthoracic needle aspiration (TTNA) is one of the primary methods for diagnosing bronchogenic carcinoma. However, across multiple studies the false negative rate for cytomorphological analysis ranges from 0.2-0.3, and among those diagnosed with lung cancer, the false positive rate for diagnosing the sub-class of small cell carcinoma averages 0.09. These false results lead to additional invasive diagnostic studies and delay treatment. In an effort to augment accuracy and utility of cytopathologic evaluation of TTNA lung samples, we measured the previously reported c-myc x E2F-1/p21WAF1/CIP1 (p21 hereafter) gene expression index in cDNA samples from 78 non-malignant lung tissues and 57 lung cancer tissues. Using the optimal cut-off value, this test correctly classified 72/78 non-malignant and 50/57 malignant samples for a correct classification rate of 90% (95% CI 83.6% - 94.3%), sensitivity of 88%, and specificity of 92%. A CDKN2C/FOSL1 test for distinguishing non-small cell lung cancer (NSCLC) from small cell lung cancer (SCLC) had a PPV of 100% with 63% sensitivity. In order to optimize the robustness and utility of these tests, we developed qPCR methods that enable simultaneous measurement of each target gene and reference gene transcript relative to a known number of internal standard competitive template (CT) molecules using two-color flurometric analysis on real-time platform. For each gene, the native template was quantified with a sequence-specific FAM-labeled probe and the CT was quantified with a sequence-specific Quasar670 labeled probe. Results for each gene thus far demonstrate excellent linearity (R2 &amp;gt; 0.99, slope 1.0 +/- 0.05), relative accuracy (variance &amp;lt; 0.2), signal-to-analyte response (1.0 +/- 0.05) and precision (CV &amp;lt; 30%) over six orders of magnitude, and reliable detection of as few as 10 molecules. Assessing both the c-myc x E2F-1/p21 index and the CDKN2C/FOSL1 test promises to augment cytomorphologic diagnosis of bronchogenic carcinoma in TTNA samples biopsy samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5541. doi:1538-7445.AM2012-5541

A novel biosensing strategy for screening G-quadruplex ligands based on graphene oxide sheets

A novel fluorescence biosensing strategy for simple, rapid and sensitive selecting quadruplex-binding ligands was reported by using graphene oxide (GO) as the fluorescence quencher. Data from transmission electron microscopy (TEM), atomic force microscopy (AFM) image, Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy demonstrated that single-layered GO sheets were successfully prepared with well dispersion in aqueous solution. The fluorescein amidite (FAM)-labeled signal probe first adsorbed onto the surface of GO through π-stacking interaction between the ring structure in the nucleobases and the hexagonal cells of GO, and the fluorescence of the dye was quenched. When the quadruplex-binding ligands were introduced, the signal probe folded to form intramolecular G-quadruplex structure, which led to the releasing of FAM-labeled signal probe from the surface of GO and the fluorescence intensity recovered. Three series of Chinese medicine monomers were investigated by the proposed method, and the flavonoids were demonstrated to be the potential quadruplex-binding ligands by fluorescence measurement and melting temperature analysis. The results indicated that this strategy offers a simple, rapid and sensitive method for screening G-quadruplex ligands and it could find wide applications in the discovery of new antitumor drugs.

Enhancing the Fluorescence Intensity of DNA Microarrays by Using Cationic Surfactants

DNA microarrays have been used as powerful tools in genomics studies and single nucleotide polymorphisms analysis. However, the fluorescence detection used in most conventional DNA microarrays is still limited by its sensitivity. The aim of this study is to use a cationic surfactant, cetyl trimethylammonium bromide (CTAB), to enhance the fluorescence intensity of 6-carboxy-fluorescene (FAM)-labeled DNA probes immobilized on a DNA microarray. We show that in the presence of CTAB the immobilized FAM-labeled DNA probes is 11-fold brighter than that without exposure to CTAB. Similarly, when we hybridize FAM-labeled DNA targets to a DNA microarray and treat the surface with CTAB solution, the fluorescence intensity shows a 26-fold increase for perfect-match DNA targets. More importantly, the contrast between perfect-match and 1-mismatch DNA is also increased from 1.3-fold to 15-fold. This method offers a simple and efficient technique to enhance the detection limit of DNA microarrays.