Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA

2021 ◽  
Author(s):  
Dana Al Sulaiman ◽  
Alfie Gatehouse ◽  
Aleksandar P. Ivanov ◽  
Joshua B. Edel ◽  
Sylvain Ladame
Keyword(s):  
Single Molecule ◽  
Dna Fragments ◽  
Cell Free Dna ◽  
Double Stranded Dna ◽  
Free Dna ◽  
Single Molecule Analysis ◽  
Potential Tool

scholarly journals Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer

EBioMedicine ◽  
2019 ◽  
Vol 41 ◽  
pp. 345-356 ◽  
Author(s):  
Xiaoyu Liu ◽  
Lingxiao Liu ◽  
Yuan Ji ◽  
Changyu Li ◽  
Tao Wei ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Mutant Cell ◽  
Dna Fragments ◽  
Cell Free Dna ◽  
Free Dna

Plasma cell-free DNA and fraction of circulating KRAS mutations as prognostic in patients with metastatic colorectal cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 490-490 ◽  
Author(s):  
David Sefrioui ◽  
Nasrin Vasseur ◽  
Richard Sesboüé ◽  
France Blanchard ◽  
Alice Oden-Gangloff ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Plasma Cell ◽  
Circulating Tumor Dna ◽  
Dna Fragments ◽  
Wild Type ◽  
Kras Mutations ◽  
Cell Free Dna ◽  
Free Dna ◽  
Tumor Dna

490 Background: It has been suggested that detection of circulating tumor DNA may be relevant in patients with metastatic colorectal cancer (mCRC). The main objective of the present study was to evaluate a method based on the TaqMan Mutation Detection Assay (TMDA) for the detection of circulating KRAS mutations in mCRC patients. Moreover, we also investigated the prognostic impact of the plasma cell-free DNA and the fraction of circulating KRAS mutations. Methods: The study was conducted from April to July 2013 and plasma samples were prospectively collected in a series of 35 mCRC patients treated with chemotherapy (CT). QIAamp Circulating Nucleic Acid kit was used for DNA extraction and Quant-iT High Sensitivity dsDNA Assay for cf-DNA quantification. Detection of circulating tumor DNA was based on the KRAS mutations detected in tumour and was performed in plasma by the castPCR Technology TMDA. Response to CT was assessed according to RECIST criteria. The results of plasma cf-DNA and level of mutant DNA fragments were correlated with response and 3-months survival. Results: We isolated and quantified plasma cf-DNA in all patients with a mean concentration of 106 ng/mL. Among them, 18 were wild-type and 17 mutated for KRAS in the tumour. Detection of circulating KRAS mutations was performed with TMDA in 23 patients (10 KRAS wild-type and 13 KRAS mutated). The sensitivity was 62% (8/13) and specificity 100% (0/10) with a level of circulating mutant DNA fragments ranging from 0 to 29%. Plasma cf-DNA and level of circulating mutant DNA were both significantly correlated with the 3-months survival (mean 36 versus 524 ng/mL, p=0.0015 and 2% versus 29%, p<0.0001). There was a non significant trend for response to CT (respectively p=0.14 and p=0.12). Conclusions: TMDA method is a simple, accurate and non-invasive tool for the detection of circulating tumor DNA. Our preliminary results also suggest that plasma cf-DNA and fraction of mutant DNA fragments could be prognostic markers in mCRC patients.

Single-Molecule Analysis of Restriction DNA Fragments Using Fluorescence Correlation Spectroscopy

1998 ◽  
Vol 260 (2) ◽  
pp. 166-172 ◽  
Author(s):  
Masataka Kinjo ◽  
Goro Nishimura ◽  
Tomiyasu Koyama ◽  
Ülo Mets ◽  
Rudolf Rigler
Keyword(s):  
Single Molecule ◽  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Dna Fragments ◽  
Fluorescence Correlation ◽  
Single Molecule Analysis

scholarly journals Ultrasensitive hybridization capture: Reliable detection of <1 copy/mL short cell-free DNA from large-volume urine samples

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247851
Author(s):  
Amy Oreskovic ◽  
Barry R. Lutz
Keyword(s):  
Large Volume ◽  
Magnetic Beads ◽  
Limit Of Detection ◽  
Extraction Methods ◽  
Urine Samples ◽  
Hybridization Method ◽  
Analytical Methodology ◽  
Cell Free Dna ◽  
Double Stranded Dna ◽  
Free Dna

Urine cell-free DNA (cfDNA) is a valuable non-invasive biomarker with broad potential clinical applications, but there is no consensus on its optimal pre-analytical methodology, including the DNA extraction step. Due to its short length (majority of fragments <100 bp) and low concentration (ng/mL), urine cfDNA is not efficiently recovered by conventional silica-based extraction methods. To maximize sensitivity of urine cfDNA assays, we developed an ultrasensitive hybridization method that uses sequence-specific oligonucleotide capture probes immobilized on magnetic beads to improve extraction of short cfDNA from large-volume urine samples. Our hybridization method recovers near 100% (95% CI: 82.6–117.6%) of target-specific DNA from 10 mL urine, independent of fragment length (25–150 bp), and has a limit of detection of ≤5 copies of double-stranded DNA (0.5 copies/mL). Pairing hybridization with an ultrashort qPCR design, we can efficiently capture and amplify fragments as short as 25 bp. Our method enables amplification of cfDNA from 10 mL urine in a single qPCR well, tolerates variation in sample composition, and effectively removes non-target DNA. Our hybridization protocol improves upon both existing silica-based urine cfDNA extraction methods and previous hybridization-based sample preparation protocols. Two key innovations contribute to the strong performance of our method: a two-probe system enabling recovery of both strands of double-stranded DNA and dual biotinylated capture probes, which ensure consistent, high recovery by facilitating optimal probe density on the bead surface, improving thermostability of the probe-bead linkage, and eliminating interference by endogenous biotin. We originally designed the hybridization method for tuberculosis diagnosis from urine cfDNA, but expect that it will be versatile across urine cfDNA targets, and may be useful for other cfDNA sample types and applications beyond cfDNA. To make our hybridization method accessible to new users, we present a detailed protocol and straightforward guidelines for designing new capture probes.

scholarly journals Cell-free DNA fragments increase transcription in human mesenchymal stem cells, activate tlr-dependent signal pathway and supress apoptosis

2012 ◽  
Vol 58 (6) ◽  
pp. 673-683 ◽  
Author(s):  
S.V. Kostyuk ◽  
E.M. Malinovskaya ◽  
A.V. Ermakov ◽  
T.D. Smirnova ◽  
L.V. Kameneva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Functional Activity ◽  
Human Mesenchymal Stem Cells ◽  
Signal Pathway ◽  
Signal Pathways ◽  
Dna Fragments ◽  
Cell Free Dna ◽  
Free Dna ◽  
Dependent Signal

Human mesenchymal stem cells (MSCs) are now widely adopted in regenerative medicine. However, many questions on the role of different signaling pathways in the regulation of stem cell (SC) functional activity within the organism remain unaswered. In damaged regions the level of cell death increases and DNA fragments from dead cells (cell-free DNA, cfDNA) are accumulated in blood. We showed that in adipose-derived MSCs exposed in vitro to cfDNA fragments the transcription level increased (the total amount of cellular RNA and the rRNA amount rose). GC-rich CfDNA fragments (GC-DNA) activated the TLR9-dependent signal pathway: the expression of TLR9 and of TLR9-signaling pathway adapter - MyD88 - was up-regulated. AT-rich DNA fragments did not increase the TLR9 expression, though, the MyD88 expression level rose. So we suggest that AT-DNA acts via some other receptors that nevertheless activate MyD88-dependent signalling in MSCs. We also showed that cfDNA fragments decreased the activity of caspase, an apoptotic enzyme. So, cfDNA can significantly influence the functional activity of MSC by activating TLR9- and MyD88-dependent signal pathways and lowering the apoptosis level.

scholarly journals Single-molecule sequencing reveals a large population of long cell-free DNA molecules in maternal plasma

2021 ◽  
Vol 118 (50) ◽  
pp. e2114937118
Author(s):  
Stephanie C. Y. Yu ◽  
Peiyong Jiang ◽  
Wenlei Peng ◽  
Suk Hang Cheng ◽  
Y. T. Tommy Cheung ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dna Analysis ◽  
Large Population ◽  
Maternal Plasma ◽  
Dna Molecules ◽  
Plasma Dna ◽  
Cell Free Dna ◽  
Dna Molecule ◽  
Free Dna ◽  
Monogenic Diseases

In the field of circulating cell-free DNA, most of the studies have focused on short DNA molecules (e.g., <500 bp). The existence of long cell-free DNA molecules has been poorly explored. In this study, we demonstrated that single-molecule real-time sequencing allowed us to detect and analyze a substantial proportion of long DNA molecules from both fetal and maternal sources in maternal plasma. Such molecules were beyond the size detection limits of short-read sequencing technologies. The proportions of long cell-free DNA molecules in maternal plasma over 500 bp were 15.5%, 19.8%, and 32.3% for the first, second, and third trimesters, respectively. The longest fetal-derived plasma DNA molecule observed was 23,635 bp. Long plasma DNA molecules demonstrated predominance of A or G 5′ fragment ends. Pregnancies with preeclampsia demonstrated a reduction in long maternal plasma DNA molecules, reduced frequencies for selected 5′ 4-mer end motifs ending with G or A, and increased frequencies for selected motifs ending with T or C. Finally, we have developed an approach that employs the analysis of methylation patterns of the series of CpG sites on a long DNA molecule for determining its tissue origin. This approach achieved an area under the curve of 0.88 in differentiating between fetal and maternal plasma DNA molecules, enabling the determination of maternal inheritance and recombination events in the fetal genome. This work opens up potential clinical utilities of long cell-free DNA analysis in maternal plasma including noninvasive prenatal testing of monogenic diseases and detection/monitoring of pregnancy-associated disorders such as preeclampsia.

scholarly journals The Length and Distribution of Plasma Cell-Free DNA Fragments in Stroke Patients

2020 ◽  
Vol 2020 ◽  
pp. 1-6 ◽  
Author(s):  
Xiaofang Cui ◽  
Shiyi Du ◽  
Houlin Liu ◽  
Ju Liu ◽  
Qingjian Wu ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Plasma Cell ◽  
Cpg Islands ◽  
Regional Distribution ◽  
Dna Fragments ◽  
Disease Group ◽  
Cell Free Dna ◽  
Healthy Group ◽  
Free Dna ◽  
Depth Analysis

A number of studies have shown that plasma cell-free DNA is closely related to the risk of stroke, but the fragmentation status of plasma cell-free DNA and its clinical application value in ischemic stroke are still unclear. In this study, 48 patients with new ischemic stroke and 20 healthy subjects were enrolled. The second-generation high-throughput sequencing technique was used to study the plasma cell-free fragment length and regional distribution of the subjects. As noted in our results, the ratio of plasma cell-free DNA fragments in the disease group was significantly greater than that of the healthy group in the 300–400 bp range; conversely for fragments at the 75–250 bp range, the ratio of plasma cell-free DNA fragments in the patient group was apparently lower than that of the healthy group. In-depth analysis of the proportion of fragments distributed on each component of the genome was carried out. Our results recorded that the plasma cell-free DNA fragments in the disease group were inclined to the EXON, CpG islands, and ALU regions in contrast to that of the healthy group. In particular, fragments within the 300–400 bp range of the disease group were enrichment in the regions of EXON, INTRON, INTERGENIC, LINE, Fragile, ALU, and CpG islands. In summary, our findings suggested that the intracellular DNA degradation profiles could be applied to distinguish the stroke group and the healthy group, which provided a theoretical basis for the clinical diagnosis and prognosis of stroke by profiling the characteristic of plasma cell-free DNA fragments.

GC-Rich DNA Fragments and Oxidized Cell-Free DNA Have Different Effects on NF-kB and NRF2 Signaling in MSC

2016 ◽  
pp. 109-112 ◽  
Author(s):  
Vasilina A. Sergeeva ◽  
Svetlana V. Kostyuk ◽  
Elizaveta S. Ershova ◽  
Elena M. Malinovskaya ◽  
Tatiana D. Smirnova ◽  
...  
Keyword(s):  
Dna Fragments ◽  
Cell Free Dna ◽  
Free Dna

scholarly journals Enrichment of fetal and maternal long cell‐free DNA fragments from maternal plasma following DNA repair

2019 ◽  
Vol 39 (2) ◽  
pp. 88-99 ◽  
Author(s):  
Joaquim S.L. Vong ◽  
Peiyong Jiang ◽  
Suk‐Hang Cheng ◽  
Wing‐Shan Lee ◽  
Jason C.H. Tsang ◽  
...  
Keyword(s):  
Dna Repair ◽  
Maternal Plasma ◽  
Dna Fragments ◽  
Cell Free Dna ◽  
Free Dna
Sign in / Sign up

Export Citation Format

Share Document