An Automatic High-Throughput Single Nucleotide Polymorphism Genotyping Approach Based on Universal Tagged Arrays and Magnetic Nanoparticles

Li, Song; Liu, Hongna; Jia, Yingying; Mou, Xianbo; Deng, Yan; Lin, Lin; Liu, Bin; He, Nongyue

doi:10.1166/jbn.2013.1568

Cited by 26 publications

(13 citation statements)

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“…Furthermore, to realize the methylation detection of a large number of clinical samples simultaneously, a high-throughput method is necessary. 16,40,41 In this study, the high-throughput detection of the FRET signal is realized by using a microwell plate reader.…”

Section: Quantitative Analysis Of Dna Methylation Levelsmentioning

confidence: 99%

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

Zhang

Liu

et al. 2015

Nanoscale

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DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.

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Section: Quantitative Analysis Of Dna Methylation Levelsmentioning

confidence: 99%

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

Zhang

Liu

et al. 2015

Nanoscale

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“…A major challenge of SNP‐based tests is the accurate genotyping of fetal SNPs in the low fetal fraction (FF; average approximately 10% at 10‐13 gestation weeks) in cfDNA extracted from maternal blood (maternal cfDNA). High‐density array chips and high‐throughput next‐generation sequencing are efficient SNP genotyping platforms, and both have shown success in this application. The use of targeted sequencing (hybridization‐based target enrichment of 5000‐8000 SNPs) and a Bayesian analysis approach successfully determined paternity in 17 clinical cases .…”

Section: Introductionmentioning

confidence: 99%

Noninvasive prenatal paternity testing by means of SNP‐based targeted sequencing

Tam¹,

Chan²,

Tsang³

et al. 2020

Prenatal Diagnosis

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Objective To develop a method for noninvasive prenatal paternity testing based on targeted sequencing of single nucleotide polymorphisms (SNPs). Method SNPs were selected based on population genetics data. Target‐SNPs in cell‐free DNA extracted from maternal blood (maternal cfDNA) were analyzed by targeted sequencing wherein target enrichment was based on multiplex amplification using QIAseq Targeted DNA Panels with Unique Molecular Identifiers. Fetal SNP genotypes were called using a novel bioinformatics algorithm, and the combined paternity indices (CPIs) and resultant paternity probabilities were calculated. Results Fetal SNP genotypes obtained from targeted sequencing of maternal cfDNA were 100% concordant with those from amniotic fluid‐derived fetal genomic DNA. From an initial panel of 356 target‐SNPs, an average of 148 were included in paternity calculations in 15 family trio cases, generating paternity probabilities of greater than 99.9999%. All paternity results were confirmed by short‐tandem‐repeat analysis. The high specificity of the methodology was validated by successful paternity discrimination between biological fathers and their siblings and by large separations between the CPIs calculated for the biological fathers and those for 60 unrelated men. Conclusion The novel method is highly effective, with substantial improvements over similar approaches in terms of reduced number of target‐SNPs, increased accuracy, and reduced costs.

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“…With the coming of the genomic and postgenomic era, nucleic acid hybridization technology has become an important tool in a variety of biomedical applications, including sample screening for single nucleotide polymorphisms, − gene expression studies, − medical diagnosis, − and food and environmental analysis. − In particular, various molecular diagnostic methods based on solid phase nucleic acid hybridization via specific oligonucleotide probes immobilized on the surface of a solid carrier to identify a target complementary sequence in solution have developed rapidly. These methods have high specificity and sensitivity because they can facilitate the removal of nontarget molecules to greatly reduce the probability of nonspecific adsorption.…”

Section: Introductionmentioning

confidence: 99%

Determination of the Binding Constant between Oligonucleotide-Coupled Magnetic Microspheres and Target DNA

et al. 2019

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Oligonucleotide-coupled magnetic microspheres (PMPs) are commonly used as solid carriers in genetic analysis to specifically recognize, capture, and manipulate target DNA. Determining the binding constant (K A ) of nucleic acid hybridization using an assay based on magnetic microspheres (MPs) and evaluating the performance of magnetic microspheres as solid carriers has significance for many applications. In this work, we established a simple doublereciprocal plot method to determine the binding constant of nucleic acid hybridization based on magnetic microspheres. The main experimental parameters were first optimized, and the binding constants between PMPs with single-stranded DNA (ssDNA) and PMPs with double-stranded DNA (dsDNA) were 1.07 ± 0.01 × 10 8 and 0.77 ± 0.02 × 10 8 M −1 , respectively, through the established double-reciprocal plot method. Oligonucleotidecoupled magnetic microspheres have the same initial effective target binding sites (A 0 ), regardless of whether the target molecules are single-stranded or double-stranded. In addition, the binding constants between PMPs with ssDNA and PMPs with dsDNA were 18.58 and 13.37%, respectively, of the binding constants measured in solution by isothermal titration calorimetry. This finding indicates that the binding constant based on solid phase hybridization is not significantly lower than that of liquid phase hybridization, even in the presence of competing target nucleic acid molecules in solution.

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An Automatic High-Throughput Single Nucleotide Polymorphism Genotyping Approach Based on Universal Tagged Arrays and Magnetic Nanoparticles

Cited by 26 publications

References 50 publications

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

Noninvasive prenatal paternity testing by means of SNP‐based targeted sequencing

Determination of the Binding Constant between Oligonucleotide-Coupled Magnetic Microspheres and Target DNA

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