Application of Nanotechnology for Sensitive Detection of Low-Abundance Single-Nucleotide Variations in Genomic DNA: A Review

Single-nucleotide polymorphisms (SNPs) are the simplest and most common type of DNA variations in the human genome. This class of attractive genetic markers, along with point mutations, have been associated with the risk of developing a wide range of diseases, including cancer, cardiovascular diseases, autoimmune diseases, and neurodegenerative diseases. Several existing methods to detect SNPs and mutations in body fluids have faced limitations. Therefore, there is a need to focus on developing noninvasive future polymerase chain reaction (PCR)–free tools to detect low-abundant SNPs in such specimens. The detection of small concentrations of SNPs in the presence of a large background of wild-type genes is the biggest hurdle. Hence, the screening and detection of SNPs need efficient and straightforward strategies. Suitable amplification methods are being explored to avoid high-throughput settings and laborious efforts. Therefore, currently, DNA sensing methods are being explored for the ultrasensitive detection of SNPs based on the concept of nanotechnology. Owing to their small size and improved surface area, nanomaterials hold the extensive capacity to be used as biosensors in the genotyping and highly sensitive recognition of single-base mismatch in the presence of incomparable wild-type DNA fragments. Different nanomaterials have been combined with imaging and sensing techniques and amplification methods to facilitate the less time-consuming and easy detection of SNPs in different diseases. This review aims to highlight some of the most recent findings on the aspects of nanotechnology-based SNP sensing methods used for the specific and ultrasensitive detection of low-concentration SNPs and rare mutations.

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Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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Detection of Single Nucleotide Polymorphisms by Fluorescence Embedded Dye SYBR Green I Based on Graphene Oxide

Frontiers in Chemistry ◽

10.3389/fchem.2021.631959 ◽

2021 ◽

Vol 9 ◽

Author(s):

Jiaoyun Xia ◽

Tong Xu ◽

Jing Qing ◽

Lihua Wang ◽

Junlong Tang

Keyword(s):

Graphene Oxide ◽

Single Nucleotide Polymorphisms ◽

Fluorescence Emission ◽

Sybr Green ◽

Sybr Green I ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Single Base ◽

Single Base Mismatch ◽

Base Mismatch

The detection of single nucleotide polymorphisms (SNPs) is of great significance in the early diagnosis of diseases and the rational use of drugs. Thus, a novel biosensor based on the quenching effect of fluorescence-embedded SYBR Green I (SG) dye and graphene oxide (GO) was introduced in this study. The probe DNA forms a double helix structure with perfectly complementary DNA (pcDNA) and 15 single-base mismatch DNA (smDNA) respectively. SG is highly intercalated with perfectly complementary dsDNA (pc-dsDNA) and exhibits strong fluorescence emission. Single-base mismatch dsDNA (SNPs) has a loose double-stranded structure and exhibits poor SG intercalation and low fluorescence sensing. At this time, the sensor still showed poor SNP discrimination. GO has a strong effect on single-stranded DNA (ssDNA), which can reduce the fluorescence response of probe DNA and eliminate background interference. And competitively combined with ssDNA in SNPs, quenching the fluorescence of SG/SNP, while the fluorescence value of pc-dsDNA was retained, increasing the signal-to-noise ratio. At this time, the sensor has obtained excellent SNP resolution. Different SNPs detect different intensities of fluorescence in the near-infrared region to evaluate the sensor's identification of SNPs. The experimental parameters such as incubation time, incubation temperature and salt concentration were optimized. Under optimal conditions, 1 nM DNA with 0–10 nM linear range and differentiate 5% SNP were achieved. The detection method does not require labeling, is low cost, simple in operation, exhibits high SNP discrimination and can be distinguished by SNP at room temperature.

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Significant Associations of lncRNA H19 Genotypes with Susceptibility to Childhood Leukemia in Taiwan

Pharmaceuticals ◽

10.3390/ph14030235 ◽

2021 ◽

Vol 14 (3) ◽

pp. 235

Author(s):

Jen-Sheng Pei ◽

Chao-Chun Chen ◽

Wen-Shin Chang ◽

Yun-Chi Wang ◽

Jaw-Chyun Chen ◽

...

Keyword(s):

Confidence Interval ◽

Childhood Leukemia ◽

Healthy Controls ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Genotypic Distribution ◽

Heterozygous Variant ◽

Significant Difference ◽

The Difference

The purpose of our study was to investigate whether genetic variations in lncRNA H19 were associated with susceptibility to childhood leukemia. Two hundred and sixty-six childhood leukemia patients and 266 healthy controls were enrolled in Taiwan, and two single nucleotide polymorphisms (SNPs), rs2839698 and rs217727, in H19 were genotyped and analyzed. There was a significant difference in the genotypic distribution of rs2839698 between patients and healthy controls (p = 0.0277). Compared to the wild-type CC genotype, the heterozygous variant CT and homozygous variant TT genotypes were associated with significantly increased risks of childhood leukemia with an adjusted odd ratio (OR) of 1.46 (95% confidence interval (CI), 1.08–2.14, p = 0.0429) and 1.94 (95%CI, 1.15–3.31, p = 0.0169), respectively (pfor tread = 0.0277). The difference in allelic frequencies between childhood leukemia patients and controls was also significant (T versus C, adjusted OR = 1.53, 95%CI, 1.13–1.79, p = 0.0077). There were no significant differences in the genotypic and allelic distributions of rs217727 between cases and controls. Interestingly, the average level of H19 rs2839698 was statistically significantly higher for patients with CT and TT genotypes than from those with the CC genotype (p < 0.0001). Our results indicate that H19 SNP rs2839698, but not rs217727, may serve as a novel susceptibility marker for childhood leukemia.

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Associations between varied susceptibilities of PfATP4 inhibitors and genotypes in Ugandan Plasmodium falciparum isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00771-21 ◽

2021 ◽

Author(s):

Oriana Kreutzfeld ◽

Stephanie A. Rasmussen ◽

Aarti A. Ramanathan ◽

Patrick K. Tumwebaze ◽

Oswald Byaruhanga ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Single Nucleotide Polymorphisms ◽

Ex Vivo ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Field Isolates ◽

Frequent Mutations ◽

Mixed Field ◽

P Type

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda from 2016-2019. Median IC 50 s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many non-synonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%) and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92 and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

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Characterisation of the melanocortin-1 receptor gene in alpaca and identification of possible markers associated with phenotypic variations in colour

Animal Production Science ◽

10.1071/an09005 ◽

2009 ◽

Vol 49 (8) ◽

pp. 675 ◽

Cited By ~ 21

Author(s):

N. L. Feeley ◽

K. A. Munyard

Keyword(s):

Mus Musculus ◽

Bos Taurus ◽

Receptor Gene ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Melanocortin 1 Receptor ◽

Mc1r Gene ◽

Pcr Products ◽

Phenotypic Variations

The aim of this study was to determine if any correlation exists between melanocortin-1 receptor (MC1R) polymorphisms and skin and fibre colour in alpacas. Primers capable of amplifying the entire alpaca MC1R gene were designed from a comparative alignment of Bos taurus and Mus musculus MC1R gene sequences. The complete MC1R gene of 41 alpacas exhibiting a range of fibre colours, and which were sourced from farms across Australia, was sequenced from PCR products. Twenty-one single nucleotide polymorphisms were identified within MC1R. Two of these polymorphisms (A82G and C901T) have the potential to reduce eumelanin production by disrupting the activity of MC1R. No agreement was observed between fibre colour alone and MC1R genotype in the 41 animals in this study. However, when the animals were assigned to groups based on the presence or absence of eumelanin in their fibre and skin, only animals that had at least one allele with the A82/C901 combination expressed eumelanin. We propose that A82/C901 is the wild-type dominant ‘E’ MC1R allele, while alpacas with either G82/T901 or G82/Y901 are homozygous for the recessive ‘e’ MC1R allele and are therefore unable to produce eumelanin.

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1202. Multimodal Sequencing of a Clonal Case Cluster of Carbapenem-Resistant Citrobacter Reveals Unexpectedly Rapid Dynamics of KPC3-Containing Plasmids

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.1035 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S364-S364

Author(s):

Roby Bhattacharyya ◽

Alejandro Pironti ◽

Bruce J Walker ◽

Abigail Manson ◽

Virginia Pierce ◽

...

Keyword(s):

Point Mutations ◽

Illumina Miseq ◽

Nucleotide Polymorphisms ◽

Sequencing Data ◽

Single Nucleotide ◽

Carbapenem Resistant ◽

Oxford Nanopore ◽

Close Relationship ◽

Long Read ◽

Carbapenem Resistant Enterobacteriaceae

Abstract Background Carbapenem-resistant Enterobacteriaceae (CRE) are a major public health threat. We report four clonally related Citrobacter freundii isolates harboring the blaKPC-3 carbapenemase in April–May 2017 that are nearly identical to a strain from 2014 at the same institution. Despite differing by ≤5 single nucleotide polymorphisms (SNPs), these isolates exhibited dramatic differences in carbapenemase plasmid architecture. Methods We sequenced four carbapenem-resistant C. freundii isolates from 2017 and compared them with an ongoing CRE surveillance project at our institution. SNPs were identified from Illumina MiSeq data aligned to a reference genome using the variant caller Pilon. Plasmids were assembled from Illumina and Oxford Nanopore sequencing data using Unicycler. Results The four 2017 isolates differed from one another by 0–5 chromosomal SNPs; two were identical. With one exception, these isolates differed by >38,000 SNPs from 25 C. freundii isolates sequenced from 2013 to 2017 at the same institution for CRE surveillance. The exception was a 2014 isolate that differed by 13–16 SNPs from each 2017 isolate, with 13 SNPs common to all four. Each C. freundii isolate harbored wild-type blaKPC-3. Despite the close relationship among the 2017 cluster, the plasmids harboring the blaKPC-3 genes differed dramatically: the carbapenemase occurred in one of the two different plasmids, with rearrangements between these plasmids across isolates. The related 2014 isolate harbored both plasmids, each with a separate copy of blaKPC-3. No transmission chains were found between any of the affected patients. Conclusion WGS confirmed clonality among four contemporaneous blaKPC-3-containing C. freundii isolates, and marked similarity with a 2014 isolate, within an institution. That only 13–16 SNPs varied between the 2014 and 2017 isolates suggests durable persistence of the blaKPC-3 gene within this lineage in a hospital ecosystem. The plasmids harboring these carbapenemase genes proved remarkably plastic, with plasmid loss and rearrangements occurring on the same time scale as two to three chromosomal point mutations. Combining short and long-read sequencing in a case cluster uniquely revealed unexpectedly rapid dynamics of carbapenemase plasmids, providing critical insight into their manner of spread. Disclosures M. J. Ferraro, SeLux Diagnostics: Scientific Advisor and Shareholder, Consulting fee. D. C. Hooper, SeLux Diagnostics: Scientific Advisor, Consulting fee.

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Multiplex, fluorescent, solid-phase minisequencing for efficient screening of DNA sequence variation

Clinical Chemistry ◽

10.1093/clinchem/42.9.1391 ◽

1996 ◽

Vol 42 (9) ◽

pp. 1391-1397 ◽

Cited By ~ 59

Author(s):

T Pastinen ◽

J Partanen ◽

A C Syvänen

Keyword(s):

Single Nucleotide Polymorphisms ◽

Solid Phase ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Dna Templates ◽

Single Nucleotide ◽

Large Numbers ◽

Dna Sequence Variation ◽

Hla Dqa1 ◽

Efficient Screening

Abstract We developed a multiplex, solid-phase minisequencing method to detect multiple single-nucleotide polymorphisms in an undivided sample. The amplified DNA templates are first captured on a manifold. Then, with multiple minisequencing primers of various sizes, single-nucleotide extension reactions are carried out simultaneously with fluorescently labeled dideoxynucleotides. The size of the extended product, determined by using a DNA sequencing instrument, defines the site of the polymorphisms, and the incorporated nucleotide gives the identity of the nucleotide at each site. HLA-DQA1 typing was used as a model system to evaluate the method. The DR2 subgroup of the HLA-DRB1 gene was typed along with the DQA1 gene to demonstrate the feasibility of the method in analyzing multiple genes at multiple sites simultaneously. The method is generally applicable for screening any single-nucleotide polymorphisms or point mutations, and its manifold format allows practical handling of large numbers of samples.

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Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

Communications Biology ◽

10.1038/s42003-019-0745-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 15

Author(s):

Hye Kyung Lee ◽

Harold E. Smith ◽

Chengyu Liu ◽

Michaela Willi ◽

Lothar Hennighausen

Keyword(s):

Point Mutations ◽

Mouse Embryos ◽

Single Nucleotide Variants ◽

Single Nucleotide ◽

Base Editing ◽

Genome Wide ◽

Wide Range ◽

Family Based ◽

Correct Point ◽

Adenine Base

AbstractDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

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simuG: a general-purpose genome simulator

Bioinformatics ◽

10.1093/bioinformatics/btz424 ◽

2019 ◽

Vol 35 (21) ◽

pp. 4442-4444 ◽

Cited By ~ 10

Author(s):

Jia-Xing Yue ◽

Gianni Liti

Keyword(s):

Copy Number Variants ◽

General Purpose ◽

Supplementary Information ◽

Nucleotide Polymorphisms ◽

Bioinformatics Analyses ◽

Full Spectrum ◽

Single Nucleotide ◽

Genomic Variants ◽

Wide Range ◽

Simulation Based

Abstract Summary Simulated genomes with pre-defined and random genomic variants can be very useful for benchmarking genomic and bioinformatics analyses. Here we introduce simuG, a lightweight tool for simulating the full-spectrum of genomic variants (single nucleotide polymorphisms, Insertions/Deletions, copy number variants, inversions and translocations) for any organisms (including human). The simplicity and versatility of simuG make it a unique general-purpose genome simulator for a wide-range of simulation-based applications. Availability and implementation Code in Perl along with user manual and testing data is available at https://github.com/yjx1217/simuG. This software is free for use under the MIT license. Supplementary information Supplementary data are available at Bioinformatics online.

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