scholarly journals High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

2019 ◽  
Author(s):  
Brandon D. Wilson ◽  
Michael Eisenstein ◽  
H. Tom Soh
Keyword(s):  
Molecular Diagnostics ◽  
Dna Sequences ◽  
Rolling Circle Amplification ◽  
Quantitative Detection ◽  
Synthesis Methods ◽  
Nanopore Sequencing ◽  
Single Nucleotide Variants ◽  
Simple Method ◽  
Lower Accuracy ◽  
Target Dna

AbstractNanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. These can be sequenced on the Oxford Nanopore Technology’s (ONT) MinION platform, and the resulting repeat sequences aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings.One Sentence SummaryWe introduce a simple method of accurately sequencing ultra-short (<100bp) target DNA on a nanopore sequencing platform.

scholarly journals Target DNA detection and quantitation on a single cell with single base resolution

TECHNOLOGY ◽  
2013 ◽  
Vol 01 (01) ◽  
pp. 88-96 ◽  
Author(s):  
Tania Konry ◽  
Adam Lerner ◽  
Martin L. Yarmush ◽  
Irina V. Smolina
Keyword(s):  
Single Cells ◽  
Rolling Circle Amplification ◽  
Copy Number Variations ◽  
New Method ◽  
Quantitative Detection ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Single Base ◽  
Target Dna ◽  
Single Base Resolution

In this report, we present a new method for sensitive detection of short DNA sites in single cells with single base resolution. The method combines peptide nucleic acid (PNA) openers as the tagging probes, together with isothermal rolling circle amplification (RCA) and fluorescence-based detection, all performed in a cells-in-flow format. Bis-PNAs provide single base resolution, while RCA ensures linear signal amplification. We applied this method to detect the oncoviral DNA inserts in cancer cell lines using a flow-cytometry system. We also demonstrated quantitative detection of the selected signature sites within single cells in microfluidic nano-liter droplets. Our results show single-nucleotide polymorphism (SNP) discrimination and detection of copy-number variations (CNV) under isothermal non-denaturing conditions. This new method is ideal for many applications in which ultra-sensitive DNA characterization with single base resolution is desired on the level of single cells.

A high sensitivity wireless mass-loading surface acoustic wave DNA biosensor

2014 ◽  
Vol 28 (07) ◽  
pp. 1450056 ◽  
Author(s):  
Hua-Lin Cai ◽  
Yi Yang ◽  
Yi-Han Zhang ◽  
Chang-Jian Zhou ◽  
Cang-Ran Guo ◽  
...  
Keyword(s):  
Acoustic Wave ◽  
Surface Acoustic Wave ◽  
Dna Sequences ◽  
Biological Treatment ◽  
High Performance ◽  
Processing System ◽  
High Sensitivity ◽  
Treatment Method ◽  
Saw Sensor ◽  
Target Dna

In this paper, a surface acoustic wave (SAW) biosensor with gold delay area on LiNbO 3 substrate detecting DNA sequences is proposed. By well-designed device parameters of the SAW sensor, it achieves a high performance for highly sensitive detection of target DNA. In addition, an effective biological treatment method for DNA immobilization and abundant experimental verification of the sensing effect have made it a reliable device in DNA detection. The loading mass of the probe and target DNA sequences is obtained from the frequency shifts, which are big enough in this work due to an effective biological treatment. The experimental results show that the biosensor has a high sensitivity of 1.2 pg/ml/Hz and high selectivity characteristic is also verified by the few responses of other substances. In combination with wireless transceiver, we develop a wireless receiving and processing system that can directly display the detection results.

Restriction of P-element insertions at the Notch locus of Drosophila melanogaster

1987 ◽  
Vol 7 (4) ◽  
pp. 1545-1548
Author(s):  
M R Kelley ◽  
S Kidd ◽  
R L Berg ◽  
M W Young
Keyword(s):  
Drosophila Melanogaster ◽  
Dna Sequences ◽  
Consensus Sequence ◽  
P Element ◽  
Induced Mutations ◽  
P Elements ◽  
Target Dna ◽  
Complex Locus ◽  
Additional Level ◽  
Derivatives Of

P elements move about the Drosophila melanogaster genome in a nonrandom fashion, preferring some chromosomal targets for insertion over others (J. C. J. Eeken, F. H. Sobels, V. Hyland, and A. P. Schalet, Mutat. Res. 150:261-275, 1985; W. R. Engels, Annu. Rev. Genet. 17:315-344, 1983; M. D. Golubovsky, Y. N. Ivanov, and M. M. Green, Proc. Natl. Acad. Sci. USA 74:2973-2975, 1977; M. J. Simmons and J. K. Lim, Proc. Natl. Acad. Sci. USA 77:6042-6046, 1980). Some of this specificity may be due to recognition of a particular DNA sequence in the target DNA; derivatives of an 8-base-pair consensus sequence are occupied by these transposable elements at many different chromosomal locations (K. O'Hare and G. M. Rubin, Cell 34:25-36, 1983). An additional level of specificity of P-element insertions is described in this paper. Of 14 mutations induced in the complex locus Notch by hybrid dysgenesis, 13 involved P-element insertions at or near the transcription start site of the gene. This clustering was not seen in other transposable element-induced mutations of Notch. DNA sequences homologous to the previously described consensus target for P-element insertion are not preferentially located in this region of the locus. The choice of a chromosomal site for integration appears to be based on more subtle variations in chromosome structure that are probably associated with activation or expression of the target gene.

Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

Direct detection of circulating microRNAs in serum of cancer patients by coupling protein-facilitated specific enrichment and rolling circle amplification

2014 ◽  
Vol 50 (25) ◽  
pp. 3292-3295 ◽  
Author(s):  
Cheng-Yi Hong ◽  
Xian Chen ◽  
Juan Li ◽  
Jing-Hua Chen ◽  
Guonan Chen ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Direct Detection ◽  
Rolling Circle Amplification ◽  
Circulating Mirnas ◽  
Simple Method ◽  
Circulating Micrornas ◽  
Rolling Circle ◽  
Coupling Protein

A simple method for direct detection of circulating miRNAs in serum by coupling p19 protein-facilitated specific enrichment and RCA.

One-Carbon Metabolism: Linking Nutritional Biochemistry to Epigenetic Programming of Long-Term Development

2019 ◽  
Vol 7 (1) ◽  
pp. 263-287 ◽  
Author(s):  
Constance E. Clare ◽  
Amey H. Brassington ◽  
Wing Yee Kwong ◽  
Kevin D. Sinclair
Keyword(s):  
Dna Sequences ◽  
Methylation Of Dna ◽  
Epigenetic Effects ◽  
Epigenetic Programming ◽  
Target Dna ◽  
Ethnic Variability ◽  
Associated Proteins ◽  
Epigenetic Gene Regulation ◽  
One Carbon Metabolism

One-carbon (1C) metabolism comprises a series of interlinking metabolic pathways that include the methionine and folate cycles that are central to cellular function, providing 1C units (methyl groups) for the synthesis of DNA, polyamines, amino acids, creatine, and phospholipids. S-adenosylmethionine is a potent aminopropyl and methyl donor within these cycles and serves as the principal substrate for methylation of DNA, associated proteins, and RNA. We propose that 1C metabolism functions as a key biochemical conduit between parental environment and epigenetic regulation of early development and that interindividual and ethnic variability in epigenetic-gene regulation arises because of genetic variants within 1C genes, associated epigenetic regulators, and differentially methylated target DNA sequences. We present evidence to support these propositions, drawing upon studies undertaken in humans and animals. We conclude that future studies should assess the epigenetic effects of cumulative (multigenerational) dietary imbalances contemporaneously in both parents, as this better represents the human experience.

scholarly journals Enhancement of target specificity of CRISPR–Cas12a by using a chimeric DNA–RNA guide

2020 ◽  
Vol 48 (15) ◽  
pp. 8601-8616 ◽  
Author(s):  
Hanseop Kim ◽  
Wi-jae Lee ◽  
Yeounsun Oh ◽  
Seung-Hun Kang ◽  
Junho K Hur ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Engineering ◽  
Target Genes ◽  
Target Sequence ◽  
Energy Potential ◽  
Guide Rna ◽  
Sequence Recognition ◽  
Protospacer Adjacent Motif ◽  
Effective Manner ◽  
Target Dna

Abstract The CRISPR–Cas9 system is widely used for target-specific genome engineering. CRISPR–Cas12a (Cpf1) is one of the CRISPR effectors that controls target genes by recognizing thymine-rich protospacer adjacent motif (PAM) sequences. Cas12a has a higher sensitivity to mismatches in the guide RNA than does Cas9; therefore, off-target sequence recognition and cleavage are lower. However, it tolerates mismatches in regions distant from the PAM sequence (TTTN or TTN) in the protospacer, and off-target cleavage issues may become more problematic when Cas12a activity is improved for therapeutic purposes. Therefore, we investigated off-target cleavage by Cas12a and modified the Cas12a (cr)RNA to address the off-target cleavage issue. We developed a CRISPR–Cas12a that can induce mutations in target DNA sequences in a highly specific and effective manner by partially substituting the (cr)RNA with DNA to change the energy potential of base pairing to the target DNA. A model to explain how chimeric (cr)RNA guided CRISPR–Cas12a and SpCas9 nickase effectively work in the intracellular genome is suggested. Chimeric guide-based CRISPR- Cas12a genome editing with reduced off-target cleavage, and the resultant, increased safety has potential for therapeutic applications in incurable diseases caused by genetic mutations.

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