scholarly journals Encapsulation of Long Genomic DNA into a Confinement of a Polyelectrolyte Microcapsule: A Single-Molecule Insight

ACS Omega ◽  
2019 ◽  
Vol 4 (1) ◽  
pp. 458-464
Author(s):  
Anatoly Zinchenko ◽  
Eisuke Inagaki ◽  
Shizuaki Murata
Keyword(s):  
Single Molecule ◽  
Genomic Dna ◽  
Polyelectrolyte Microcapsule

scholarly journals Analysis of HLA-G long-read genomic sequences in mother–offspring pairs with preeclampsia

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ayako Nishizawa ◽  
Kazuki Kumada ◽  
Keiko Tateno ◽  
Maiko Wagata ◽  
Sakae Saito ◽  
...  
Keyword(s):  
Single Molecule ◽  
Gene Polymorphisms ◽  
Genomic Dna ◽  
Genomic Sequences ◽  
Genomic Sequencing ◽  
Public Database ◽  
Coding Sequences ◽  
Pacbio Rs Ii ◽  
Potential Association ◽  
Long Read

AbstractPreeclampsia is a pregnancy-induced disorder that is characterized by hypertension and is a leading cause of perinatal and maternal–fetal morbidity and mortality. HLA-G is thought to play important roles in maternal–fetal immune tolerance, and the associations between HLA-G gene polymorphisms and the onset of pregnancy-related diseases have been explored extensively. Because contiguous genomic sequencing is difficult, the association between the HLA-G genotype and preeclampsia onset is controversial. In this study, genomic sequences of the HLA-G region (5.2 kb) from 31 pairs of mother–offspring genomic DNA samples (18 pairs from normal pregnancies/births and 13 from preeclampsia births) were obtained by single-molecule real-time sequencing using the PacBio RS II platform. The HLA-G alleles identified in our cohort matched seven known HLA-G alleles, but we also identified two new HLA-G alleles at the fourth-field resolution and compared them with nucleotide sequences from a public database that consisted of coding sequences that cover the 3.1-kb HLA-G gene span. Intriguingly, a potential association between preeclampsia onset and the poly T stretch within the downstream region of the HLA-G*01:01:01:01 allele was found. Our study suggests that long-read sequencing of HLA-G will provide clues for characterizing HLA-G variants that are involved in the pathophysiology of preeclampsia.

Feasibility of Population Scale Comprehensive Identification and Analysis of Complex Structural Variations in Cancer Genome Using Nanochannel Array

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 476-476
Author(s):  
Alex Hastie ◽  
Ernest Lam ◽  
Heng Dai ◽  
Warren Andrews ◽  
Andy Pang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Complex Traits ◽  
Genomic Dna ◽  
Genomic Sequence ◽  
Conflicts Of Interest ◽  
Large Population ◽  
Sequence Motif ◽  
Structural Variations ◽  
Hematological Cancers ◽  
Population Scale

Abstract Diseases with complex traits such as hematological cancer are known to be associated with large structural variations (SV > 1 kb). Techniques such as karyotyping, FISH and aCGH have been common tools for gross chromosomal lesion analysis. Yet population scale comprehensive SV analysis with these tools remains impractical, tedious or incomplete such as missing balanced lesions by aCGH. Recently, high throughput Next Generation Sequencing (NGS) has generated a large amount of sequencing reads that have rapidly reduced costs and are effective in detection of SNPs and small indels; however, complicated by the fact that large structural variations, often spanning tens to hundreds of thousands of base pairs or involving complex rearrangements throughout the genome, are hard for current short read sequencing technology to assemble or infer. Therefore, there is a blind spot in effectively detecting SVs within this range (1 kb ~ 1 Mb) due to insufficient tools. Here we demonstrate a technology that rapidly linearizes very long strands of genomic DNA (100 kb to Mbs) through NanoChannels to directly visualize large SVs and rearrangements preserved within intact genomic DNA at the single molecule level. Through specific sequence motif labeling, de novo genome physical maps are assembled within hours and hundreds to thousands of SV events, balanced or imbalanced, are called with no a priori knowledge of the samples. Preliminary testing data from a group of cancer samples and multiple trio families will be shown to demonstrate this highly comprehensive and cost effective approach, with results validated by direct single-molecule images and multiple orthogonal methods. We also show long spanning molecules would provide very valuable information of precise mapping of viral genomic sequence integration in human genomes that are potentially associated with malignant cell transformation. For the first time, it is now feasible to do large population-based comprehensive genome structural variation studies on a single platform. This innovations will transform the biomedical research, diagnosis and treatment of hematological cancers that result from structural variations and chromosomal lesions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Single-molecule imaging reveals a common mechanism shared by G-quadruplex–resolving helicases

2016 ◽  
Vol 113 (30) ◽  
pp. 8448-8453 ◽  
Author(s):  
Ramreddy Tippana ◽  
Helen Hwang ◽  
Patricia L. Opresko ◽  
Vilhelm A. Bohr ◽  
Sua Myong
Keyword(s):  
Secondary Structure ◽  
Substrate Specificity ◽  
Single Molecule ◽  
Genomic Dna ◽  
Rna Helicase ◽  
Resolving Power ◽  
Common Mechanism ◽  
Dna Molecule ◽  
G Quadruplex ◽  
Stable Formation

G-quadruplex (GQ) is a four stranded DNA secondary structure that arises from a guanine rich sequence. Stable formation of GQ in genomic DNA can be counteracted by the resolving activity of specialized helicases including RNA helicase AU (associated with AU rich elements) (RHAU) (G4 resolvase 1), Bloom helicase (BLM), and Werner helicase (WRN). However, their substrate specificity and the mechanism involved in GQ unfolding remain uncertain. Here, we report that RHAU, BLM, and WRN exhibit distinct GQ conformation specificity, but use a common mechanism of repetitive unfolding that leads to disrupting GQ structure multiple times in succession. Such unfolding activity of RHAU leads to efficient annealing exclusively within the same DNA molecule. The same resolving activity is sufficient to dislodge a stably bound GQ ligand, including BRACO-19, NMM, and Phen-DC3. Our study demonstrates a plausible biological scheme where different helicases are delegated to resolve specific GQ structures by using a common repetitive unfolding mechanism that provides a robust resolving power.

Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection

2010 ◽  
Vol 402 (1) ◽  
pp. 83-90 ◽  
Author(s):  
Ekaterina Protozanova ◽  
Meng Zhang ◽  
Eric J. White ◽  
Emilia T. Mollova ◽  
Dirk Ten Broeck ◽  
...  
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
Genomic Dna ◽  
Single Molecule Detection ◽  
High Resolution Mapping ◽  
Resolution Mapping

Single-molecule micromanipulation studies of DNA and architectural proteins

2008 ◽  
Vol 36 (4) ◽  
pp. 732-737 ◽  
Author(s):  
Remus Th. Dame
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Genomic Dna ◽  
Dna Binding Proteins ◽  
Binding Kinetics ◽  
Sequence Specificity ◽  
Effective Volume ◽  
Dna Complexes ◽  
Ensemble Techniques ◽  
Architectural Proteins

Architectural proteins play a key role in the folding, organization and compaction of genomic DNA in all organisms. By bending, bridging or wrapping DNA, these proteins ensure that its effective volume is reduced sufficiently to fit inside the cell or a dedicated cellular organelle, the nucleus (in bacteria/archaea and in eukaryotes respectively). In addition, the properties of many of these proteins permit them to play specific roles as architectural cofactors in a large variety of DNA transactions. However, as architectural proteins often bind DNA with low sequence specificity and affinity, it is hard to investigate their interaction using biochemical ensemble techniques. Single-molecule micromanipulation approaches that probe the properties of DNA-binding proteins by pulling on individual protein–DNA complexes have, in this respect, proved to be a very powerful alternative. Besides revealing architectural properties, these approaches can also reveal unique parameters not accessible to biochemical approaches, such as the binding kinetics and unbinding forces of individual proteins.

scholarly journals Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation

2016 ◽  
Vol 113 (16) ◽  
pp. 4338-4343 ◽  
Author(s):  
Chun-Xiao Song ◽  
Jiajie Diao ◽  
Axel T. Brunger ◽  
Stephen R. Quake
Keyword(s):  
Single Molecule ◽  
Genomic Dna ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mammalian Genome ◽  
Single Molecule Fluorescence ◽  
Dna Molecule ◽  
Cpg Sites ◽  
Mammalian Genomes ◽  
Mouse Genomic

The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.

Direct Detection of Bacterial Genomic DNA at Sub-Femtomolar Concentrations Using Single Molecule Arrays

2013 ◽  
Vol 85 (3) ◽  
pp. 1932-1939 ◽  
Author(s):  
Linan Song ◽  
Dandan Shan ◽  
Mingwei Zhao ◽  
Brian A. Pink ◽  
Kaitlin A. Minnehan ◽  
...  
Keyword(s):  
Single Molecule ◽  
Genomic Dna ◽  
Direct Detection

Cytosine Variant Calling with High-throughput Nanopore Sequencing

2016 ◽  
Author(s):  
Arthur C. Rand ◽  
Miten Jain ◽  
Jordan Eizenga ◽  
Audrey Musselman-Brown ◽  
Hugh E. Olsen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Dirichlet Process ◽  
Genomic Dna ◽  
Probabilistic Method ◽  
Simultaneous Detection ◽  
Ionic Current ◽  
Variant Calling ◽  
Variable Order ◽  
Chemical Modifications ◽  
Oxford Nanopore

AbstractChemical modifications to DNA regulate cellular state and function. The Oxford Nanopore MinION is a portable single-molecule DNA sequencer that can sequence long fragments of genomic DNA. Here we show that the MinION can be used to detect and map two chemical modifications cytosine, 5-methylcytosine and 5-hydroxymethylcytosine. We present a probabilistic method that enables expansion of the nucleotide alphabet to include bases containing chemical modifications. Our results on synthetic DNA show that individual cytosine base modifications can be classified with accuracy up to 95% in a three-way comparison and 98% in a two-way comparison.Statement of SignificanceNanopore-based sequencing technology can produce long reads from unamplified genomic DNA, potentially allowing the characterization of chemical modifications and non-canonical DNA nucleotides as they occur in the cell. As the throughput of nanopore sequencers improves, simultaneous detection of multiple epigenetic modifications to cytosines will become an important capability of these devices. Here we present a statistical model that allows the Oxford Nanopore Technologies MinION to be used for detecting chemical modifications to cytosine using standard DNA preparation and sequencing techniques. Our method is based on modeling the ionic current due to DNA k-mers with a variable-order hidden Markov model where the emissions are distributed according to a hierarchical Dirichlet process mixture of normal distributions. This method provides a principled way to expand the nucleotide alphabet to allow for variant calling of modified bases.

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