scholarly journals Massively parallel single-molecule telomere length measurement with digital real-time PCR

2020 ◽  
Vol 6 (34) ◽  
pp. eabb7944 ◽  
Author(s):  
Yongqiang Luo ◽  
Ramya Viswanathan ◽  
Manoor Prakash Hande ◽  
Amos Hong Pheng Loh ◽  
Lih Feng Cheow
Keyword(s):  
Telomere Length ◽  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Length Distribution ◽  
Telomere Maintenance ◽  
Massively Parallel ◽  
Primary Tumors ◽  
Absolute Length

Telomere length is a promising biomarker for age-associated diseases and cancer, but there are still substantial challenges to routine telomere analysis in clinics because of the lack of a simple and rapid yet scalable method for measurement. We developed the single telomere absolute-length rapid (STAR) assay, a novel high-throughput digital real-time PCR approach for rapidly measuring the absolute lengths and quantities of individual telomere molecules. We show that this technique provides the accuracy and sensitivity to uncover associations between telomere length distribution and telomere maintenance mechanisms in cancer cell lines and primary tumors. The results indicate that the STAR assay is a powerful tool to enable the use of telomere length distribution as a biomarker in disease and population-wide studies.

Nanoscale “Curtain Rods”: High-Throughput Tools for Studying DNA-Protein Interactions

2008 ◽  
Vol 1138 ◽  
Author(s):  
Teresa Fazio ◽  
Mari-Liis Visnapuu ◽  
Shalom J. Wind ◽  
Eric Greene
Keyword(s):  
Data Acquisition ◽  
Real Time ◽  
Lipid Bilayer ◽  
High Throughput ◽  
Single Molecule ◽  
Protein Interactions ◽  
Massively Parallel ◽  
Dna Interactions ◽  
Parallel Data ◽  
Protein Dna Interactions

AbstractIn this work, we combine nanoscale engineering with single-molecule biology to probe the biochemical interactions between individual proteins and DNA. This approach, a vast improvement over previous methods, constructs a platform to observe thousands of protein-DNA interactions in real time with unprecedented detail. A key challenge in these experiments involves collecting enough statistically relevant data in order to analyze reactions which are designed to be probed individually. “DNA curtains” are formed by flowing the DNA tethered to a lipid bilayer across nanopatterned barriers, facilitating massively parallel data acquisition.

Highly Sensitive Detection of Paraquat with Pillar[5]arene as an aptamer in α-Hemolysin Nanopore

2021 ◽  
Author(s):  
Xiaojia Jiang ◽  
Mingsong Zang ◽  
Fei Li ◽  
Chunxi Hou ◽  
Quan Luo ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Single Molecule ◽  
Single Molecule Detection ◽  
Sensitive Detection ◽  
High Throughput Analysis ◽  
Throughput Analysis ◽  
The Real ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

Biological nanopore-based techniques have attracted more and more attention recently in the field of single-molecule detection, because they allow the real-time, sensitive, high-throughput analysis. Herein, we report an engineered biological...

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

scholarly journals Increased Expression of the Pro-Protein Convertase Furin Predicts Decreased Survival in Ovarian Cancer

2007 ◽  
Vol 29 (4) ◽  
pp. 289-299
Author(s):  
Robert E. Page ◽  
Andrés J. P. Klein-Szanto ◽  
Samuel Litwin ◽  
Emmanuelle Nicolas ◽  
Raid Al-Jumaily ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Tumor Progression ◽  
Real Time ◽  
Cell Lines ◽  
Cancer Cell ◽  
Real Time Pcr ◽  
Ovarian Tumor ◽  
Cancer Cell Lines ◽  
Rt Pcr ◽  
Primary Tumors

Background: Proprotein convertases (PCs) are serine proteases that after restricted proteolysis activate many proteins that play a crucial role in cancer such as metalloproteinases, growth factors and growth factor receptors, adhesion molecules, and angiogenic factors. Although the expression of several PCs is increased in many tumors, their expression in primary ovarian tumors has not been studied in detail. We sought to determine if there was an association between the expression of the ubiquitously expressed PCs, furin, PACE-4, PC-5 and PC-7, and ovarian tumor progression. Methods: We assessed their expression by RT-PCR, Real-time PCR, Western blot, and immunohistochemistry using cells derived from normal human ovarian surface epithelium (HOSE) and cancer cell lines as well as ovarian epithelial cancer specimens (45 RT-PCR/Real-time PCR, and 120 archival specimens for Immunohistochemistry). Results: We found that furin expression was restricted to the cancer cell lines. In contrast, PACE-4 and PC-7 showed expression only in normal HOSE cells lines. Furthermore, furin was predominantly expressed in primary tumors from patients who survived for less than five years. The other PCs are either expressed in the group of survivors (PC-7 and PACE4) or expressed in low amounts (PC-5). Conclusions: Our studies point to a clear relationship between furin and ovarian cancer. In addition, these results show that furin exhibits the closest association with ovarian cancer among the ubiquitously expressed PCs, arguing against the redundancy of these proteases. In summary, furin may constitute a marker for ovarian tumor progression and could contribute to predict the outcome of this disease.

scholarly journals The association of male infertility with telomere length: A case control study

2021 ◽  
Vol 8 (4) ◽  
pp. 325-332
Author(s):  
Kate Deepali Rajesh ◽  
Puranam Vatsalaswamy ◽  
Manvikar Purshotam Rao
Keyword(s):  
Male Infertility ◽  
Telomere Length ◽  
Real Time ◽  
Real Time Pcr ◽  
Case Control Study ◽  
Case Control ◽  
Control Group ◽  
Significant Difference ◽  
Control Study ◽  
Sperm Telomere Length

To study the relevance of sperm telomere length and infertility in men. : Our case-control study included twenty-five males in couple with sub-fertility/infertility (test group) and twenty five healthy males (control group) with proven paternity in the age group 25 to 35 years. The Absolute Sperm Telomere length (aSTL) was measured by real-time PCR. We investigated whether any significant difference in the aSTL value existed between the groups and analysed the relationship between aSTL and other sperm parameters.The mean (SE) aSTL recorded in the infertile cases was significantly shorter than for the control group being 140.60 (6.66) Kb/genome and 239.63 (12.32) Kb/genome respectively (p <0.001) A weak correlation was eminent between aSTL kb/genome and the total sperm count mil/ml (rho= 0.04, p - 0.86), progressive sperm motility (rho= - 0.02, p=0.934) and sperm viability (rho= - 0.07 p=0.741) in the infertile group. The measurement of aSTL by real-time PCR is a simple and rapid method that offers further paramount information with respective to the quality of sperm. It is befitted for epidemiological studies, hence opening new perspectives in the evaluation of male infertility. Limitations - Our study was confined to men aged between 25 and 35 years. Further comparative studies are needed to explore the significance of STL and infertility in older males. Additional studies will help illumine the significance of aSTL as a prognostic biomarker in assisted reproduction.

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2021 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

scholarly journals Estimating Clinically Relevant Cut-Off Values for a High-Throughput Quantitative Real-Time PCR Detecting Bacterial Respiratory Pathogens in Cattle

2021 ◽  
Vol 8 ◽  
Author(s):  
Alicia F. Klompmaker ◽  
Maria Brydensholt ◽  
Anne Marie Michelsen ◽  
Matthew J. Denwood ◽  
Carsten T. Kirkeby ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Age Groups ◽  
Therapeutic Interventions ◽  
Respiratory Pathogens ◽  
Logistic Regression Models ◽  
Histophilus Somni ◽  
Nasal Swabs

Bovine respiratory disease (BRD) results from interactions between pathogens, environmental stressors, and host factors. Obtaining a diagnosis of the causal pathogens is challenging but the use of high-throughput real-time PCR (rtPCR) may help target preventive and therapeutic interventions. The aim of this study was to improve the interpretation of rtPCR results by analysing their associations with clinical observations. The objective was to develop and illustrate a field-data driven statistical method to guide the selection of relevant quantification cycle cut-off values for pathogens associated with BRD for the high-throughput rtPCR system “Fluidigm BioMark HD” based on nasal swabs from calves. We used data from 36 herds enrolled in a Danish field study where 340 calves within pre-determined age-groups were subject to clinical examination and nasal swabs up to four times. The samples were analysed with the rtPCR system. Each of the 1,025 observation units were classified as sick with BRD or healthy, based on clinical scores. The optimal rtPCR results to predict BRD were investigated for Pasteurella multocida, Mycoplasma bovis, Histophilus somni, Mannheimia haemolytica, and Trueperella pyogenes by interpreting scatterplots and results of mixed effects logistic regression models. The clinically relevant rtPCR cut-off suggested for P. multocida and M. bovis was ≤ 21.3. For H. somni it was ≤ 17.4, while no cut-off could be determined for M. haemolytica and T. pyogenes. The demonstrated approach can provide objective support in the choice of clinically relevant cut-offs. However, for robust performance of the regression model sufficient amounts of suitable data are required.

scholarly journals Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

1999 ◽  
Vol 37 (6) ◽  
pp. 1941-1947 ◽  
Author(s):  
Alexander J. Ryncarz ◽  
James Goddard ◽  
Anna Wald ◽  
Meei-Li Huang ◽  
Bernard Roizman ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Genital Tract ◽  
Clinical Samples ◽  
Pcr Assay ◽  
The Mean ◽  
Simplex Virus

We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results obtained by assays with monoclonal antibodies against HSV-1 or HSV-2. Thus, while the real-time PCR is slightly less sensitive than the gel-based liquid hybridization system, the high throughput, the lack of contamination during processing, the better reproducibility, and the better ability to type the isolates rapidly make the real-time PCR a valuable tool for clinical investigation and diagnosis of HSV infection.

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