Accurate genotyping of fragmented DNA using a toehold assisted padlock probe

2021 ◽  
Vol 179 ◽  
pp. 113079
Author(s):  
Yanmin Gao ◽  
Hongyan Qiao ◽  
Victor Pan ◽  
Zhaoguan Wang ◽  
Jiaojiao Li ◽  
...  
Keyword(s):  
Padlock Probe ◽  
Fragmented Dna

scholarly journals MiRNA Detection Using a Rolling Circle Amplification and RNA-Cutting Allosteric Deoxyribozyme Dual Signal Amplification Strategy

Biosensors ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 222
Author(s):  
Chenxin Fang ◽  
Ping Ouyang ◽  
Yuxing Yang ◽  
Yang Qing ◽  
Jialun Han ◽  
...  
Keyword(s):  
Signal Amplification ◽  
Rolling Circle Amplification ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Sensing Platform ◽  
Mirna Detection ◽  
Substrate Cleavage ◽  
Amplification Strategy ◽  
Circular Template ◽  
Dual Signal Amplification

A microRNA (miRNA) detection platform composed of a rolling circle amplification (RCA) system and an allosteric deoxyribozyme system is proposed, which can detect miRNA-21 rapidly and efficiently. Padlock probe hybridization with the target miRNA is achieved through complementary base pairing and the padlock probe forms a closed circular template under the action of ligase; this circular template results in RCA. In the presence of DNA polymerase, RCA proceeds and a long chain with numerous repeating units is formed. In the presence of single-stranded DNA (H1 and H2), multi-component nucleic acid enzymes (MNAzymes) are formed that have the ability to cleave substrates. Finally, substrates containing fluorescent and quenching groups and magnesium ions are added to the system to activate the MNAzyme and the substrate cleavage reaction, thus achieving fluorescence intensity amplification. The RCA–MNAzyme system has dual signal amplification and presents a sensing platform that demonstrates broad prospects in the analysis and detection of nucleic acids.

scholarly journals Correlation between the DNA fragmentation index (DFI) and sperm morphology of infertile patients

2021 ◽  
Author(s):  
Alberto Ferrigno ◽  
Giovanni Ruvolo ◽  
Giuseppina Capra ◽  
Nicola Serra ◽  
Liana Bosco
Keyword(s):  
Dna Fragmentation ◽  
Sperm Morphology ◽  
Seminal Fluid ◽  
Image Analyzer ◽  
Fragmented Dna ◽  
Fragmentation Index ◽  
Group A ◽  
Group B ◽  
Infertile Patients ◽  
Dna Fragmentation Index

Abstract Purpose To evaluate the correlation between the DNA Fragmentation Index (DFI) and sperm morphology in patients undergoing ICSI, as a predictive parameter in reproductive outcomes. Methods A retrospective study was conducted on 125 infertile patients enrolled in a fertility clinic. Seminal characteristics were measured following the WHO guidelines (2010) for the examination of the seminal fluid. After collecting motile sperm population by pellet swim up, DFI was calculated and simultaneously associated with sperm morphology using in situ TUNEL assay and an image analyzer software in at least 250 spermatozoa for each patient. Results All subjects were divided into two groups according to a cutoff established, by choice, of the sperm DFI (15%): group A (< 15%) consisting of 65 patients and group B (≥ 15%) of 60 patients. Data were analyzed using non-parametric statistical methods. The results demonstrate that there is no statistical difference between the two groups in seminal characteristics. The collective data show a high significant correlation, suggesting that spermatozoa with abnormal morphology are the best candidates to contain DNA damage (p < 0.001). Also, when group A is compared with group B, an increased percentage of morphologically normal spermatozoa with fragmented DNA was observed in patients, with DFI values ≥ 15% (p < 0.001). Conclusion These results are aimed at providing an exact value of DFI in morphologically normal spermatozoa, which will be helpful to the embryologist in evaluating the risk of transferring, during the ICSI procedure, a spermatozoon whit normal morphology but fragmented DNA.

The assessment of sperm DNA fragmentation in the saltwater crocodile (Crocodylus porosus)

2017 ◽  
Vol 29 (3) ◽  
pp. 630 ◽  
Author(s):  
S. D. Johnston ◽  
C. López-Fernández ◽  
F. Arroyo ◽  
J. L. Fernández ◽  
J. Gosálvez
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmented Dna ◽  
Sperm Dna ◽  
Saltwater Crocodile ◽  
Crocodylus Porosus ◽  
Dispersion Test ◽  
Sperm Nuclei

Herein we report a method of assessing DNA fragmentation in the saltwater crocodile using the sperm chromatin dispersion test (SCDt) after including frozen–thawed spermatozoa in a microgel (Halomax; Halotech DNA, Madrid, Spain). Following controlled protein depletion, which included a reducing agent, sperm nuclei with fragmented DNA showed a homogeneous and larger halo of chromatin dispersion with a corresponding reduced nucleoid core compared with sperm with non-fragmented DNA. The presence of DNA damage was confirmed directly by incorporation of modified nucleotides using in situ nick translation (ISNT) and indirectly by studying the correlation of the SCDt with the results of DNA damage visualisation using a two-tailed comet assay (r = 0.90; P = 0.037). Results of the SCDt immediately following thawing and after 5 h incubation at 37°C in order to induce a range of DNA damage revealed individual crocodile differences in both the baseline level of DNA damage and DNA longevity.

Fragmented DNA transport in dendrites of retinal neurons during apoptotic cell death

2004 ◽  
Vol 1007 (1-2) ◽  
pp. 183-187 ◽  
Author(s):  
Akira Hara ◽  
Masayuki Niwa ◽  
Masako Kumada ◽  
Nami Kitaori ◽  
Tetsuya Yamamoto ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Retinal Neurons ◽  
Dna Transport ◽  
Fragmented Dna

scholarly journals Sperm DNA fragmentation in men of different age

2019 ◽  
Vol 20 (4) ◽  
pp. 39-44
Author(s):  
S. Sh. Khayat ◽  
E. E. Bragina ◽  
E. A. Arifulin ◽  
E. M. Lazareva ◽  
T. M. Sorokina ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Count ◽  
Semen Analysis ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Dna Fragmentation ◽  
Dna Breaks ◽  
Study Objective ◽  
Fragmented Dna ◽  
Group 2 ◽  
Group 1

The study objective is to analyze the content of spermatozoa with single and double-stranded DNA breaks in different age groups.Materials and methods. The level of DNA fragmentation was studied in 300 ejaculate samples obtained from 266 sub- or infertile men. The group 1 included 150 samples obtained from 131 patients under the age of 45 (21–44 years), the group 2 included 150 samples obtained from 135 patients above the age of 45 (45–68 years). Mean ages were 34.8 ± 3.9 and 48.6 ± 3.1 years, respectively. The number of sperm with fragmented DNA was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method on ejaculate smears. The number of spermatozoa with >15 % of fragmented DNA was considered elevated. Standard semen analysis was performed in 117 and 97 men from the groups 1 and 2, respectively.Results. The number of sperm with fragmented DNA varied in ejaculated samples from 1.5 to 64.5 %. Mean number of sperm with DNA breaks in the group 1 (12.0 ± 6.0 %) was significantly lower than in the group 2 (16.1 ± 8.3 %, p <0.05). Mean sperm count in the ejaculate of the group 1 (267.0 ± 198.7 million) was significantly higher than in the group 2 (201.0 ± 162.9 million, p = 0.02).Conclusion. We revealed that in men over the age of 45 years, the percentage of spermatozoa with DNA fragmentation is higher than in men under 45 years of age, it may indirectly indicate an increased level of reactive oxygen species in the seminal plasma in older patients. 

scholarly journals Molecular detection of human papillomavirus (HPV) in highly fragmented DNA from cervical cancer biopsies using double-nested PCR

MethodsX ◽  
2018 ◽  
Vol 5 ◽  
pp. 569-578 ◽  
Author(s):  
Leabaneng Tawe ◽  
Surbhi Grover ◽  
Mohan Narasimhamurthy ◽  
Sikhulile Moyo ◽  
Simani Gaseitsiwe ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Human Papillomavirus ◽  
Nested Pcr ◽  
Molecular Detection ◽  
Fragmented Dna

Corneocyte lysis and fragmented DNA considerations for the cellular component of forensic touch DNA

2021 ◽  
Vol 51 ◽  
pp. 102428
Author(s):  
Julia Burrill ◽  
Elli Rammenou ◽  
Fatima Alawar ◽  
Barbara Daniel ◽  
Nunzianda Frascione
Keyword(s):  
Cellular Component ◽  
Touch Dna ◽  
Fragmented Dna

scholarly journals Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza

2018 ◽  
Vol 64 (12) ◽  
pp. 1704-1712 ◽  
Author(s):  
Felix Neumann ◽  
Iván Hernández-Neuta ◽  
Malin Grabbe ◽  
Narayanan Madaboosi ◽  
Jan Albert ◽  
...  
Keyword(s):  
Seasonal Influenza ◽  
Rolling Circle Amplification ◽  
High Specificity ◽  
Clinical Samples ◽  
Padlock Probe ◽  
Rolling Circle ◽  
Diagnostic Assays ◽  
Padlock Probes ◽  
Probe Assay ◽  
Digital Quantification

Abstract BACKGROUND Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants—A(H1N1), A(H3N2), B/Yamagata, and B/Victoria—and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa™ Flu A/B & RSV Direct. RESULTS The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital readout for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

scholarly journals In Situ Detection and Quantification of AR-V7, AR-FL, PSA, and KRAS Point Mutations in Circulating Tumor Cells

2018 ◽  
Vol 64 (3) ◽  
pp. 536-546 ◽  
Author(s):  
Amin El-Heliebi ◽  
Claudia Hille ◽  
Navya Laxman ◽  
Jessica Svedlund ◽  
Christoph Haudum ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Specific Antigen ◽  
Padlock Probe ◽  
Castration Resistant Prostate Cancer ◽  
Kras Mutations ◽  
Padlock Probes ◽  
Kras Status

Abstract BACKGROUND Liquid biopsies can be used in castration-resistant prostate cancer (CRPC) to detect androgen receptor splice variant 7 (AR-V7), a splicing product of the androgen receptor. Patients with AR-V7-positive circulating tumor cells (CTCs) have greater benefit of taxane chemotherapy compared with novel hormonal therapies, indicating a treatment-selection biomarker. Likewise, in those with pancreatic cancer (PaCa), KRAS mutations act as prognostic biomarkers. Thus, there is an urgent need for technology investigating the expression and mutation status of CTCs. Here, we report an approach that adds AR-V7 or KRAS status to CTC enumeration, compatible with multiple CTC-isolation platforms. METHODS We studied 3 independent CTC-isolation devices (CellCollector, Parsortix, CellSearch) for the evaluation of AR-V7 or KRAS status of CTCs with in situ padlock probe technology. Padlock probes allow highly specific detection and visualization of transcripts on a cellular level. We applied padlock probes for detecting AR-V7, androgen receptor full length (AR-FL), and prostate-specific antigen (PSA) in CRPC and KRAS wild-type (wt) and mutant (mut) transcripts in PaCa in CTCs from 46 patients. RESULTS In situ analysis showed that 71% (22 of 31) of CRPC patients had detectable AR-V7 expression ranging from low to high expression [1–76 rolling circle products (RCPs)/CTC]. In PaCa patients, 40% (6 of 15) had KRAS mut expressing CTCs with 1 to 8 RCPs/CTC. In situ padlock probe analysis revealed CTCs with no detectable cytokeratin expression but positivity for AR-V7 or KRAS mut transcripts. CONCLUSIONS Padlock probe technology enables quantification of AR-V7, AR-FL, PSA, and KRAS mut/wt transcripts in CTCs. The technology is easily applicable in routine laboratories and compatible with multiple CTC-isolation devices.

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