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1
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Jeddi F, Faghfuri E, Mehranfar S, Soozangar N. The common bisulfite-conversion-based techniques to analyze DNA methylation in human cancers. Cancer Cell Int 2024; 24:240. [PMID: 38982390 PMCID: PMC11234524 DOI: 10.1186/s12935-024-03405-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2024] [Accepted: 06/11/2024] [Indexed: 07/11/2024] Open
Abstract
DNA methylation is an important molecular modification that plays a key role in the expression of cancer genes. Evaluation of epigenetic changes, hypomethylation and hypermethylation, in specific genes are applied for cancer diagnosis. Numerous studies have concentrated on describing DNA methylation patterns as biomarkers for cancer diagnosis monitoring and predicting response to cancer therapy. Various techniques for detecting DNA methylation status in cancers are based on sodium bisulfite treatment. According to the application of these methods in research and clinical studies, they have a number of advantages and disadvantages. The current review highlights sodium bisulfite treatment-based techniques, as well as, the advantages, drawbacks, and applications of these methods in the evaluation of human cancers.
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Affiliation(s)
- Farhad Jeddi
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
- Department of Genetics and Pathology, School of Medicine, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Elnaz Faghfuri
- Digestive Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran
| | - Sahar Mehranfar
- Department of Genetics and Immunology, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran
| | - Narges Soozangar
- Zoonoses Research Center, Ardabil University of Medical Sciences, Ardabil, Iran.
- Digestive Diseases Research Center, Ardabil University of Medical Sciences, Ardabil, Iran.
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2
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Li C, Jia H, Wei X, Xue G, Xu J, Cheng R, Cheng Y, Song Q, Shen Z, Xue C. Single-Nucleotide-Specific Lipidic Nanoflares for Precise and Visible Detection of KRAS Mutations via Toehold-Initiated Self-Priming DNA Polymerization. Anal Chem 2024; 96:4205-4212. [PMID: 38433457 DOI: 10.1021/acs.analchem.3c05511] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/05/2024]
Abstract
Accurate identification of single-nucleotide mutations in circulating tumor DNA (ctDNA) is critical for cancer surveillance and cell biology research. However, achieving precise and sensitive detection of ctDNAs in complex physiological environments remains challenging due to their low expression and interference from numerous homologous species. This study introduces single-nucleotide-specific lipidic nanoflares designed for the precise and visible detection of ctDNA via toehold-initiated self-priming DNA polymerization (TPP). This system can be assembled from only a single cholesterol-conjugated multifunctional molecular beacon (MMB) via hydrophobicity-mediated aggregation. This results in a compact, high-density, and nick-hidden arrangement of MMBs on the surface of lipidic micelles, thereby enhancing their biostability and localized concentrations. The assay commences with the binding of frequently mutated regions of ctDNA to the MMB toehold domain. This domain is the proximal holding point for initiating the TPP-based strand-displacement reaction, which is the key step in enabling the discrimination of single-base mutations. We successfully detected a single-base mutation in ctDNA (KRAS G12D) in its wild-type gene (KRAS WT), which is one of the most frequently mutated ctDNAs. Notably, coexisting homologous species did not interfere with signal transduction, and small differences in these variations can be visualized by fluorescence imaging. The limit of detection was as low as 10 amol, with the system functioning well in physiological media. In particular, this system allowed us to resolve genetic mutations in the KRAS gene in colorectal cancer, suggesting its high potential in clinical diagnosis and personalized medicine.
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Affiliation(s)
- Chan Li
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Haiyan Jia
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Xiaoling Wei
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Guohui Xue
- Department of Clinical Laboratory, Jiujiang No. 1 People's Hospital, Jiujiang 332000, Jiangxi, PR China
| | - Jianguo Xu
- Key Laboratory of Molecular Recognition and Sensing, College of Biological, Chemical Sciences and Engineering, Jiaxing University, Jiaxing 314001, PR China
| | - Ruize Cheng
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Yinghao Cheng
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Qiufeng Song
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Zhifa Shen
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
| | - Chang Xue
- Wenzhou Key Laboratory of Cancer Pathogenesis and Translation, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325000, PR China
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3
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Krishnaswamy S. S. Paul Bajaj (1944-2022). J Thromb Haemost 2023; 21:2640-2641. [PMID: 37479539 DOI: 10.1016/j.jtha.2023.06.025] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2023] [Accepted: 06/21/2023] [Indexed: 07/23/2023]
Affiliation(s)
- Sriram Krishnaswamy
- Division of Hematology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Pennsylvania, USA.
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4
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Ortiz M, Jauset-Rubio M, Trummer O, Foessl I, Kodr D, Acero JL, Botero ML, Biggs P, Lenartowicz D, Trajanoska K, Rivadeneira F, Hocek M, Obermayer-Pietsch B, O’Sullivan CK. Generic Platform for the Multiplexed Targeted Electrochemical Detection of Osteoporosis-Associated Single Nucleotide Polymorphisms Using Recombinase Polymerase Solid-Phase Primer Elongation and Ferrocene-Modified Nucleoside Triphosphates. ACS CENTRAL SCIENCE 2023; 9:1591-1602. [PMID: 37637735 PMCID: PMC10450878 DOI: 10.1021/acscentsci.3c00243] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/27/2023] [Indexed: 08/29/2023]
Abstract
Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.
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Affiliation(s)
- Mayreli Ortiz
- INTERFIBIO
Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain
| | - Miriam Jauset-Rubio
- INTERFIBIO
Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain
| | - Olivia Trummer
- Division
of Endocrinology and Diabetology, Department of Internal Medicine, Medical University of Graz, 8036 Graz, Austria
| | - Ines Foessl
- Division
of Endocrinology and Diabetology, Department of Internal Medicine, Medical University of Graz, 8036 Graz, Austria
| | - David Kodr
- Institute
of Organic Chemistry and Biochemistry, Czech
Academy of Sciences, Flemingovo namesti 2, CZ 16610 Prague 6, Czech Republic
| | - Josep Lluís Acero
- INTERFIBIO
Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain
| | - Mary Luz Botero
- INTERFIBIO
Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain
| | - Phil Biggs
- Labman
Automation
Ltd., Seamer Hill, Stokesley, North Yorkshire, TS9 5NQ U.K.
| | - Daniel Lenartowicz
- Labman
Automation
Ltd., Seamer Hill, Stokesley, North Yorkshire, TS9 5NQ U.K.
| | - Katerina Trajanoska
- Department
of Internal Medicine, Erasmus MC, 40 3015 Rotterdam, The Netherlands
| | | | - Michal Hocek
- Institute
of Organic Chemistry and Biochemistry, Czech
Academy of Sciences, Flemingovo namesti 2, CZ 16610 Prague 6, Czech Republic
- Department
of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, CZ-12843 Prague 2, Czech Republic
| | - Barbara Obermayer-Pietsch
- Division
of Endocrinology and Diabetology, Department of Internal Medicine, Medical University of Graz, 8036 Graz, Austria
| | - Ciara K. O’Sullivan
- INTERFIBIO
Research Group, Departament d’Enginyeria Química, Universitat Rovira i Virgili, 43007 Tarragona, Spain
- Institució
Catalana de Recerca i Estudis Avancats (ICREA), 08010 Barcelona, Spain
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5
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Solid-phase recombinase polymerase amplification using ferrocene-labelled dNTPs for electrochemical detection of single nucleotide polymorphisms. Biosens Bioelectron 2022; 198:113825. [PMID: 34838372 DOI: 10.1016/j.bios.2021.113825] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/11/2021] [Revised: 11/01/2021] [Accepted: 11/18/2021] [Indexed: 11/21/2022]
Abstract
Hypertrophic cardiomyopathies (HCM) are the principal cause of sudden cardiac death in young athletes and it is estimated that 1 in 500 people have HCM. The aim of this work was to develop an electrochemical platform for the detection of HCM-associated SNP in the Myosin Heavy Chain 7 (MYH7) gene, in fingerprick blood samples. The platform exploits isothermal solid-phase primer elongation using recombinase polymerase amplification with either individual or a combination of four ferrocene-labelled nucleoside triphosphates. Four thiolated reverse primers containing a variable base at their 3' end were immobilised on individual gold electrodes of an array. Following hybridisation with target DNA, solid phase recombinase polymerase amplification was carried out and primer elongation incorporating the ferrocene labelled oligonucleotides was only detected at one of the electrodes, thus facilitating identification of the SNP under interrogation. The assay was applied to the direct detection of the SNP in fingerprick blood samples from eight different individuals, with the results obtained corroborating with next generation sequencing. The ability to be able to robustly identify the SNP using a 10 μL fingerprick sample, demonstrates that SNP discrimination is achieved using low femtomolar (ca. 8 × 105 copies DNA) levels of DNA.
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Ortiz M, Jauset-Rubio M, Skouridou V, Machado D, Viveiros M, Clark TG, Simonova A, Kodr D, Hocek M, O’Sullivan CK. Electrochemical Detection of Single-Nucleotide Polymorphism Associated with Rifampicin Resistance in Mycobacterium tuberculosis Using Solid-Phase Primer Elongation with Ferrocene-Linked Redox-Labeled Nucleotides. ACS Sens 2021; 6:4398-4407. [PMID: 34797987 PMCID: PMC8715531 DOI: 10.1021/acssensors.1c01710] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
![]()
Here, we report the
electrochemical detection of single-point mutations
using solid-phase isothermal primer elongation with redox-labeled
oligonucleotides. A single-base mutation associated with resistance
to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system
to demonstrate a proof-of-concept of the approach. Four 5′-thiolated
primers, designed to be complementary with the same fragment of the
target sequence and differing only in the last base, addressing the
polymorphic site, were self-assembled via chemisorption on individual
gold electrodes of an array. Following hybridization with single-stranded
DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled
2′-deoxyribonucleoside triphosphates (dNFcTPs) was
only observed to proceed at the electrode where there was full complementarity
between the surface-tethered probe and the target DNA being interrogated.
We tested all four ferrocenylethynyl-linked dNTPs and optimized the
ratio of labeled/natural nucleotides to achieve maximum sensitivity.
Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated
primer elongation at 37 °C for 5 min was optimal for the enzymatic
incorporation of a ferrocene-labeled nucleotide, achieving unequivocal
electrochemical detection of a single-point mutation in 14 samples
of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can
be extended to multiplexed electrochemical single-point mutation genotyping.
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Affiliation(s)
- Mayreli Ortiz
- Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain
| | - Miriam Jauset-Rubio
- Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain
| | - Vasso Skouridou
- Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain
| | - Diana Machado
- Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisbon, Portugal
| | - Miguel Viveiros
- Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisbon, Portugal
| | - Taane G. Clark
- Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, Rua da Junqueira, 100, 1349-008 Lisbon, Portugal
- Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, WC1E 7HT London, U.K
| | - Anna Simonova
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam.2, 16610 Prague 6, Czech Republic
- Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, 12843 Prague 2, Czech Republic
| | - David Kodr
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam.2, 16610 Prague 6, Czech Republic
| | - Michal Hocek
- Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nam.2, 16610 Prague 6, Czech Republic
- Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova 8, 12843 Prague 2, Czech Republic
| | - Ciara K. O’Sullivan
- Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda Països Catalans 26, 43007 Tarragona, Spain
- Institució Catalana de Recerca i Estudis Avançats, Passeig Lluis Companys 23, 08010 Barcelona, Spain
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7
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Breyer JP, Smith JR. Practical genotyping by single-nucleotide primer extension. Biol Methods Protoc 2020; 5:bpaa002. [PMID: 32382659 PMCID: PMC7200932 DOI: 10.1093/biomethods/bpaa002] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 01/17/2020] [Accepted: 01/27/2020] [Indexed: 11/20/2022] Open
Abstract
Genome-wide association studies bring into focus specific genetic variants of particular interest for which validation is often sought in large numbers of study subjects. Practical alternative methods are limiting for the application of genotyping few variants in many samples. A common scenario is the need to genotype a study population at a specific high-value single nucleotide polymorphism (SNP) or insertion-deletion (indel). Not all such variants, however, will be amenable to assay by a given approach. We have adapted a single-nucleotide primer extension (SNuPE) method that may be tailored to genotype a required variant, and implemented it as a useful general laboratory protocol. We demonstrate reliable application for production-scale genotyping, successfully converting 87% of SNPs and indels for assay with an estimated error rate of 0.003. Our implementation of the SNuPE genotyping assay is a viable addition to existing alternative methods; it is readily customizable, scalable, and uses standard reagents and a laboratory plate reader.
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Affiliation(s)
- Joan P Breyer
- Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN, USA
| | - Jeffrey R Smith
- Division of Genetic Medicine, Department of Medicine, Vanderbilt Genetics Institute, Vanderbilt University Medical Center, Nashville, TN, USA.,Medical Research Service, VA Tennessee Valley Healthcare System, Nashville, TN, USA
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8
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Haselmann V, Ahmad-Nejad P, Geilenkeuser WJ, Duda A, Gabor M, Eichner R, Patton S, Neumaier M. Results of the first external quality assessment scheme (EQA) for isolation and analysis of circulating tumour DNA (ctDNA). Clin Chem Lab Med 2019; 56:220-228. [PMID: 28841569 DOI: 10.1515/cclm-2017-0283] [Citation(s) in RCA: 44] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2017] [Accepted: 07/25/2017] [Indexed: 12/18/2022]
Abstract
BACKGROUND Circulating tumour DNA (ctDNA) is considered to have a high potential for future management of malignancies. This pilot external quality assessment (EQA) scheme aimed to address issues of analytical quality in this new area of laboratory diagnostics. METHODS The EQA scheme consisted of three 2-mL EDTA-plasma samples spiked with fragmented genomic DNA with a mutant allele frequency ranging from 0% to 10% dedicated to the analysis of nine known sequence variations in KRAS codon 12/13 and of BRAF V600E. Laboratories reported: (1) time elapsed for processing, (2) storage temperatures, (3) methods for extraction and quantification, (4) genotyping methodologies and (5) results. RESULTS Specimens were sent to 42 laboratories from 10 European countries; 72.3% reported to isolate cell-free DNA (cfDNA) manually, 62.5% used the entire plasma volume for cfDNA isolation and 38.5% used >10% of cfDNA extracted for downstream genotyping. Of the methods used for quantification, PicoGreen demonstrated the lowest coefficient of variation (33.7%). For genotyping, 11 different methods were reported with the highest error rate observed for Sanger sequencing and the lowest for highly sensitive approaches like digital PCR. In total, 197 genotypes were determined with an overall error rate of 6.09%. CONCLUSIONS This pilot EQA scheme illustrates the current variability in multiple phases of cfDNA processing and analysis of ctDNA resulting in an overall error rate of 6.09%. The areas with the greatest variance and clinical impact included specimen volume, cfDNA quantification method, and preference of genotyping platform. Regarding quality assurance, there is an urgent need for harmonisation of procedures and workflows.
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Affiliation(s)
- Verena Haselmann
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Parviz Ahmad-Nejad
- Institute for Medical Laboratory Diagnostics, Centre for Clinical and Translational Research (CCTR), HELIOS Hospital, Witten/Herdecke University, Wuppertal, Germany
| | - Wolf J Geilenkeuser
- Reference-Institute for Bioanalytics, German Society for Clinical Chemistry and Laboratory Medicine (DGKL), Bonn, Germany
| | - Angelika Duda
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Merle Gabor
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Romy Eichner
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
| | - Simon Patton
- European Molecular Genetic Quality Network (EMQN), Manchester Centre for Genomic Medicine, St Mary's Hospital, Manchester, UK
| | - Michael Neumaier
- Institute for Clinical Chemistry, Medical Faculty Mannheim of the University of Heidelberg, University Hospital Mannheim, Mannheim, Germany
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9
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Wu S, Liang P, Yu H, Xu X, Liu Y, Lou X, Xiao Y. Amplified single base-pair mismatch detection via aggregation of exonuclease-sheared gold nanoparticles. Anal Chem 2014; 86:3461-7. [PMID: 24611947 PMCID: PMC3982981 DOI: 10.1021/ac4040373] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
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Single
nucleotide polymorphism (SNP) detection is important for
early diagnosis, clinical prognostics, and disease prevention, and
a rapid and sensitive low-cost SNP detection assay would be valuable
for resource-limited clinical settings. We present a simple platform
that enables sensitive, naked-eye detection of SNPs with minimal reagent
and equipment requirements at room temperature within 15 min. SNP
detection is performed in a single tube with one set of DNA probe-modified
gold nanoparticles (AuNPs), a single exonuclease (Exo III), and the
target in question. Exo III’s apurinic endonucleolytic activity
differentially processes hybrid duplexes between the AuNP-bound probe
and DNA targets that are perfectly matched or contain a single-base
mismatch. For perfectly matched targets, Exo III’s exonuclease
activity facilitates a process of target recycling that rapidly shears
DNA probes from the particles, generating an AuNP aggregation-induced
color change, whereas no such change occurs for mismatched targets.
This color change is easily observed with as little as 2 nM of target,
100-fold lower than the target concentration required for reliable
naked eye observation with unmodified AuNPs in well-optimized reaction
conditions. We further demonstrate that this system can effectively
discriminate a range of different mismatches.
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Affiliation(s)
- Shuo Wu
- Department of Chemistry and Biochemistry, Florida International University , 11200 SW eighth Street, Miami, FL, 33199
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10
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Seow N, Lai PS, Yung LYL. Gold nanostructures for the multiplex detection of glucose-6-phosphate dehydrogenase gene mutations. Anal Biochem 2014; 451:56-62. [PMID: 24491445 DOI: 10.1016/j.ab.2014.01.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2013] [Revised: 01/18/2014] [Accepted: 01/21/2014] [Indexed: 10/25/2022]
Abstract
We describe a gold nanoparticle-based technique for the detection of single-base mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene, a condition that can lead to neonatal jaundice and hemolytic anemia. The aim of this technique is to clearly distinguish different mutations frequently described within the Asian population from their wild-type counterparts and across different mutant variants. Gold nanoparticles of different sizes were synthesized, and each was conjugated with a single-strand DNA (ssDNA) sequence specific for a particular mutation in the G6PD gene. It was found that only mutant targets presented a characteristic band on the agarose gel, indicating the successful formation of dimeric nanostructures. No such dimer bands were observed for the wild-type targets. The difference in the relative dimer band levels allowed different mutant variants to be distinguished from one another. The technique was further validated using G6PD-deficient patient samples. This simple mutation detection method with direct result readout is amenable for rapid and mass screening of samples.
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Affiliation(s)
- Nianjia Seow
- Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore 119260, Singapore
| | - Poh San Lai
- Department of Pediatrics, National University Health System, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228, Singapore.
| | - Lin-Yue Lanry Yung
- Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore 119260, Singapore.
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11
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Kristensen LS, Treppendahl MB, Grønbæk K. Analysis of epigenetic modifications of DNA in human cells. ACTA ACUST UNITED AC 2013; Chapter 20:Unit20.2. [PMID: 23595599 DOI: 10.1002/0471142905.hg2002s77] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells. DNA methylation and hydroxymethylation are epigenetic modifications found in human cells, which are deeply implicated in normal cellular processes as well as in several major human diseases. Here, a range of different methods for the analyses of DNA methylation and hydroxymethylation at locus-specific and genome-wide scales is described.
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12
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Van K, Kang YJ, Shim SR, Lee SH. Genome-wide scan of the soybean genome using degenerate oligonucleotide primed PCR: an example for studying large complex genome structure. Genes Genomics 2012. [DOI: 10.1007/s13258-011-0238-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
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13
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Akkinepalli H, Ereful N, Liu Y, Malabanan K, Howells R, Stamati K, Powell W, Leung H, Greenland A, Mackay I, Lee D. Snapshots of gene expression in rice: limitations for allelic expression imbalance determination. Genome 2012; 55:400-6. [DOI: 10.1139/g2012-023] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
In an initial investigation of differential expression of genes caused by cis-acting regulatory elements in rice, the lack of reproducibility led us to question the basic premise of allelic expression imbalance determination: namely that departures of cDNA expression ratios from those observed in genomic DNA provide unequivocal evidence of cis-acting polymorphisms. This paper describes experiments designed to demonstrate that stochastic variation in low copy number of targets in PCR reactions give variable allelic ratios even when starting with the same copy numbers of the two alleles. These significant departures from an expected 1:1 ratio provide an explanation to the lack of reproducibility observed for our cDNA measurements.
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Affiliation(s)
- Harika Akkinepalli
- John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
| | - Nelzo Ereful
- Plant Molecular Genetics Lab, Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Los Banos, the Philippines
| | - Yan Liu
- Plant Molecular Genetics Lab, Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Los Banos, the Philippines
| | - Kaye Malabanan
- Plant Molecular Genetics Lab, Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Los Banos, the Philippines
| | - Rhian Howells
- John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
| | - Konstantina Stamati
- John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
| | - Wayne Powell
- Institute of Biological, Environmental & Rural Sciences, Aberystwyth University, Penglais, Aberystwyth, Ceredigion, UK
| | - Hei Leung
- Plant Molecular Genetics Lab, Plant Breeding, Genetics and Biotechnology, International Rice Research Institute, Los Banos, the Philippines
| | - Andy Greenland
- John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
| | - Ian Mackay
- John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
| | - David Lee
- John Bingham Laboratory, National Institute of Agricultural Botany, Huntingdon Road, Cambridge CB3 0LE, UK
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Mikeska T, Candiloro ILM, Dobrovic A. The implications of heterogeneous DNA methylation for the accurate quantification of methylation. Epigenomics 2012; 2:561-73. [PMID: 22121974 DOI: 10.2217/epi.10.32] [Citation(s) in RCA: 114] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
DNA methylation based biomarkers have considerable potential for molecular diagnostics, both as tumor specific biomarkers for the early detection or post-therapeutic monitoring of cancer as well as prognostic and predictive biomarkers for therapeutic stratification. Particularly in the former, the accurate estimation of DNA methylation is of compelling importance. However, quantification of DNA methylation has many traps for the unwary, especially when heterogeneous methylation comprising multiple alleles with varied DNA methylation patterns (epialleles) is present. The frequent occurrence of heterogeneous methylation as distinct from a simple mixture of fully methylated and unmethylated alleles is generally not taken into account when DNA methylation is considered as a cancer biomarker. When heterogeneous DNA methylation is present, the proportion of methylated molecules is difficult to quantify without a method that allows the measurement of individual epialleles. In this article, we critically assess the methodologies frequently used to investigate DNA methylation, with an emphasis on the detection and measurement of heterogeneous DNA methylation. The adoption of digital approaches will enable the effective use of heterogeneous DNA methylation as a cancer biomarker.
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Affiliation(s)
- Thomas Mikeska
- Molecular Pathology Research & Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia.
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Harrison A, Parle-McDermott A. DNA methylation: a timeline of methods and applications. Front Genet 2011; 2:74. [PMID: 22303369 PMCID: PMC3268627 DOI: 10.3389/fgene.2011.00074] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2011] [Accepted: 10/04/2011] [Indexed: 12/20/2022] Open
Abstract
DNA methylation is a biochemical process where a DNA base, usually cytosine, is enzymatically methylated at the 5-carbon position. An epigenetic modification associated with gene regulation, DNA methylation is of paramount importance to biological health and disease. Recently, the quest to unravel the Human Epigenome commenced, calling for a modernization of previous DNA methylation profiling techniques. Here, we describe the major developments in the methodologies used over the past three decades to examine the elusive epigenome (or methylome). The earliest techniques were based on the separation of methylated and unmethylated cytosines via chromatography. The following years would see molecular techniques being employed to indirectly examine DNA methylation levels at both a genome-wide and locus-specific context, notably immunoprecipitation via anti-5'methylcytosine and selective digestion with methylation-sensitive restriction endonucleases. With the advent of sodium bisulfite treatment of DNA, a deamination reaction that converts cytosine to uracil only when unmethylated, the epigenetic modification can now be identified in the same manner as a DNA base-pair change. More recently, these three techniques have been applied to more technically advanced systems such as DNA microarrays and next-generation sequencing platforms, bringing us closer to unveiling a complete human epigenetic profile.
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Affiliation(s)
- Alan Harrison
- Nutritional Genomics Group, School of Biotechnology, Dublin City University Dublin, Ireland
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Tan K, Kajino K, Momose S, Masaoka A, Sasahara K, Shiomi K, Izumi H, Abe M, Ohtsuji N, Wang T, Hino O, Fujii H. Mesothelin (MSLN) promoter is hypomethylated in malignant mesothelioma, but its expression is not associated with methylation status of the promoter. Hum Pathol 2010; 41:1330-8. [PMID: 20573372 DOI: 10.1016/j.humpath.2010.03.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/06/2009] [Revised: 03/08/2010] [Accepted: 03/11/2010] [Indexed: 11/16/2022]
Abstract
Gene methylation leads to malignant progression in some tumors. The mechanism by which mesothelin is expressed in malignant mesothelioma (MM) is not well understood. MM is histologically divided into 3 subtypes, that is, the epithelioid, sarcomatoid, and biphasic types, and it was shown that mesothelin expression was restricted to the epithelioid type and the epithelioid component of the biphasic type of MM. However, its regulatory mechanism of expression has not been clarified. Here, we studied the expression of mesothelin by immunohistochemistry along with the methylation status of 20 CpG sites in the promoter of the mesothelin gene (MSLN) in 118 lung specimens, including 39 MM, 41 lung carcinoma, 26 nonneoplastic pulmonary lesions, and 12 normal lung tissue samples by the methylation-sensitive single nucleotide primer extension technique. We confirmed that mesothelin was expressed in the epithelioid type and epithelioid component of the biphasic type of MM but neither in the sarcomatoid type nor sarcomatous component of the biphasic type. Surprisingly, the MSLN promoter was significantly hypomethylated in the MM cases regardless of its subtype, compared with the other pulmonary lesions and normal lung tissue samples. These findings suggested that hypomethylation of the MSLN promoter may be specifically associated with the formation of MM, regardless of its expression status, and that the expression of mesothelin protein was lost in the sarcomatoid type by some unknown posttranscriptional regulatory mechanism. We also identified 4 CpG sites, among the 20 sites studied, to be more specifically hypomethylated in MM cases.
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Affiliation(s)
- Ke Tan
- Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo 113-8421, Japan
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17
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Identification and Functional Analysis of Regulatory Polymorphisms. Clin Rev Bone Miner Metab 2010. [DOI: 10.1007/s12018-009-9067-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Roberts CH, Mayor NP, Madrigal JA, Marsh SGE. Short template amplicon and multiplex megaprimer-enabled relay (STAMMER) sequencing, a simultaneous approach to higher throughput sequence-based typing of polymorphic genes. Immunogenetics 2010; 62:253-60. [PMID: 20204613 DOI: 10.1007/s00251-010-0432-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2009] [Accepted: 02/05/2010] [Indexed: 11/28/2022]
Abstract
Sequence-based typing (SBT) is a powerful method of genotyping in highly polymorphic gene systems. In standard SBT methods, both strands of a double-stranded template amplicon are sequenced in separate reactions in order to achieve high quality data across the region of interest. The amount of informative data that is obtained from the second strand sequence is often low, whilst the impact of performing second strand sequencing on costs and throughput are significant. Here we present short template amplicon and multiplex megaprimer-enabled relay (STAMMER) sequencing, a novel simultaneous sequence-based typing methodology that allows the detection of any practical amount of useful sequence from a plurality of distinct polymerase chain reaction products in a single sequencing reaction. In addition to simultaneous bidirectional sequencing, we show how the STAMMER approach can be used to simultaneously sequence a number of regions of interest that are not physically linked within the range of a single sequencing reaction. The efficiencies of this method could impact significantly on the output of SBT laboratories.
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Ferri L, Perrin E, Campana S, Tabacchioni S, Taccetti G, Cocchi P, Ravenni N, Dalmastri C, Chiarini L, Bevivino A, Manno G, Mentasti M, Fani R. Application of multiplex single nucleotide primer extension (mSNuPE) to the identification of bacteria: the Burkholderia cepacia complex case. J Microbiol Methods 2010; 80:251-6. [PMID: 20079386 DOI: 10.1016/j.mimet.2010.01.008] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2009] [Accepted: 01/06/2010] [Indexed: 10/20/2022]
Abstract
Burkholderia cepacia complex (BCC) is characterized by a complex taxonomy constituted by seventeen closely related species of both biotechnological and clinical importance. Several molecular methods have been developed to accurately identify BCC species but simpler and effective strategies for BCC classification are still needed. A single nucleotide primer extension (SNuPE) assay using gyrB as a target gene was developed to identify bacteria belonging to the B. cepacia (BCC) complex. This technique allows the successful detection and distinction of single nucleotide polymorphisms (SNPs) and is effectively applied in routine medical diagnosis since it permits to analyze routinely many samples in a few times. Seven SNuPE primers were designed analyzing the conserved regions of the BCC gyrB sequences currently available in databases. The specificity of the assay was evaluated using reference strains of some BCC species. Data obtained enabled to discriminate bacteria belonging to the species B. multivorans, B. cenocepacia (including bacteria belonging to recA lineages III-A, III-C, and III-D), B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina and B. pyrrocinia. Conversely, identification failed for B. cepacia, B. cenocepacia III-B and B. stabilis. This study demonstrates the efficacy of SNuPE technique for the identification of bacteria characterized by a complex taxonomical organization as BCC bacteria.
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Affiliation(s)
- L Ferri
- Department of Evolutionary Biology, University of Florence, Via Romana 17-19, I-50125 Florence, Italy
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Affiliation(s)
- Tomasz K Wojdacz
- Human Genetics Institute, University of Aarhus, Wilhelm Meyers Alle 240, DK-8000 Aarhus C, Denmark
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21
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Hoque MO. DNA methylation changes in prostate cancer: current developments and future clinical implementation. Expert Rev Mol Diagn 2009; 9:243-57. [PMID: 19379083 DOI: 10.1586/erm.09.10] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Promoter hypermethylation is associated with the loss of expression of tumor-suppressor genes in cancer. Currently, several genome-wide technologies are available and have been utilized to examine the extent of DNA methylation in discovery-based studies involving several physiological and disease states. Although early in the process, aberrant DNA methylation is gaining strength in the fields of cancer risk assessment, diagnosis and therapy monitoring in different cancer types. There is a need to improve existing methods for early diagnosis of prostate cancer and to identify men at risk for developing aggressive disease. Because of the ubiquity of DNA methylation changes and the ability to detect methylated DNA in several body fluids (e.g., blood and urine), this specifically altered DNA may serve, on one hand, as a possible new screening marker for prostate cancer and, on the other hand, as a tool for therapy monitoring in patients having had neoplastic disease of the prostate. Since many prostate cancer patients present with advanced disease and some present with nonspecific elevation of prostate-specific antigen without prostate cancer, early detection with high specificity and sensitivity is considered to be one of the most important approaches to reduce mortality and unwanted tension of the men with high prostate-specific antigen. Therefore, an effective screening test would have substantial clinical benefits. Furthermore, methylation markers of risk of progression of disease in patients having prostate cancer permits immediate commencement of specific treatment regimens and probably longer survival and better quality of life. This review illustrates the current benefits and limitations of potentially useful prostate cancer methylation markers that have considerable existing data and touches upon other future markers as well as the field of methylation in prostate cancer.
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Affiliation(s)
- Mohammad Obaidul Hoque
- Department of Otolaryngology and Head and Neck Surgery, The Johns Hopkins University School of Medicine, 1550 Orleans Street, CRB II, 5M.07, Baltimore, MD 21231, USA.
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Kristensen LS, Hansen LL. PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem 2009; 55:1471-83. [PMID: 19520761 DOI: 10.1373/clinchem.2008.121962] [Citation(s) in RCA: 154] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND DNA methylation is a highly characterized epigenetic modification of the human genome that is implicated in cancer. The altered DNA methylation patterns found in cancer cells include not only global hypomethylation but also discrete hypermethylation of specific genes. In particular, numerous tumor suppressor genes undergo epigenetic silencing because of hypermethylated promoter regions. Some of these genes are considered promising DNA methylation biomarkers for early cancer diagnostics, and some have been shown to be valuable for predicting prognosis or the response to therapy. CONTENT PCR-based methods that use sodium bisulfite-treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci. A number of new methods, such as methylation-specific fluorescent amplicon generation (MS-FLAG), methylation-sensitive high-resolution melting (MS-HRM), and sensitive melting analysis after real-time methylation-specific PCR (SMART-MSP), now complement the traditional PCR-based methods and promise to be valuable diagnostic tools. In particular, the HRM technique shows great potential as a diagnostic tool because of its closed-tube format and cost-effectiveness. SUMMARY Numerous traditional and new PCR-based methods have been developed for detecting DNA methylation at single loci. All have characteristic advantages and disadvantages, particularly with regard to use in clinical settings.
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The single-nucleotide primer extension (SNuPE) method for the multiplex detection of various DNA sequences: from detection of point mutations to microbial ecology. Biochem Soc Trans 2009; 37:454-9. [PMID: 19290881 DOI: 10.1042/bst0370454] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Methods based on SNuPE (single-nucleotide primer extension) have become invaluable tools for the rapid and highly specific detection of point mutations and single-nucleotide polymorphisms in the field of human genetics. In the primer extension reaction, a DNA polymerase is used to label a specific primer hybridized to the target sequence by incorporating a single labelled ddNTP (dideoxynucleotide). This labelling provides not only information about the complementary nucleotide of interest in the opposite strand but also a semiquantitative analysis of the sequence targeted by the primer. Since several subdisciplines of microbiology increasingly require cultivation-independent molecular screening tools for elucidating differences between either strains or community structures based on sequence variations of marker genes, SNuPE offers a promising alternative to the existing tool box. The present review describes the method in detail and reports the state-of-the-art applications of this technique both in the field of nucleic acid detections in human genetics and in microbiology.
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Somatic mutations in SQSTM1 detected in affected tissues from patients with sporadic Paget's disease of bone. J Bone Miner Res 2009; 24:484-94. [PMID: 19016598 PMCID: PMC2659521 DOI: 10.1359/jbmr.081105] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Paget's disease of bone (PDB) is a focal disorder of bone remodeling that leads to overgrowth of affected bone, with rare progression to osteosarcoma. Extensive studies of familial PDB showed that a majority of cases harbor germline mutations in the Sequestosome1 gene (SQSTM1). In contrast, little is known about the mutational status of SQSTM1 in sporadic PDB. We hypothesized that somatic SQSTM1 mutations might occur in the affected tissues of sporadic PDB and pagetic osteosarcoma. We used laser capture microdissection to capture homogeneous populations of cells from the affected bone or tumor of patients with sporadic PDB or pagetic osteosarcoma, respectively. DNA from these samples and appropriate controls was used for sequence analysis and allelic discrimination analysis. Two of five patients with sporadic PDB had SQSTM1(C1215T) mutations detected in their affected bone but not in their blood samples, indicating a somatic origin of the mutations. Samples from three of five sporadic pagetic osteosarcoma patients had the SQSTM1(C1215T) mutation, whereas the normal adjacent tissue from two of these tumors clearly lacked the mutation, again indicating an occurrence of somatic events. No SQSTM1 mutations were found in primary adolescent osteosarcomas. The discovery of somatic SQSTM1 mutations in sporadic PDB and pagetic osteosarcoma shows a role for SQSTM1 in both sporadic and inherited PDB. The discovery of somatically acquired mutations in both the diseased bone and tumor samples suggests a paradigm shift in our understanding of this disease.
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Wang Y, Zhang D, Zheng W, Luo J, Bai Y, Lu Z. Multiple gene methylation of nonsmall cell lung cancers evaluated with 3-dimensional microarray. Cancer 2008; 112:1325-36. [DOI: 10.1002/cncr.23312] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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Van K, Onoda S, Kim MY, Kim KD, Lee SH. Allelic variation of the Waxy gene in foxtail millet [Setaria italica (L.) P. Beauv.] by single nucleotide polymorphisms. Mol Genet Genomics 2007; 279:255-66. [PMID: 18157676 DOI: 10.1007/s00438-007-0310-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2007] [Accepted: 11/28/2007] [Indexed: 11/29/2022]
Abstract
The Waxy (Wx) gene product controls the formation of a straight chain polymer of amylose in the starch pathway. Dominance/recessiveness of the Wx allele is associated with amylose content, leading to non-waxy/waxy phenotypes. For a total of 113 foxtail millet accessions, agronomic traits and the molecular differences of the Wx gene were surveyed to evaluate genetic diversities. Molecular types were associated with phenotypes determined by four specific primer sets (non-waxy, Type I; low amylose, Type VI; waxy, Type IV or V). Additionally, the insertion of transposable element in waxy was confirmed by ex1/TSI2R, TSI2F/ex2, ex2int2/TSI7R and TSI7F/ex4r. Seventeen single nucleotide polymorphims (SNPs) were observed from non-coding regions, while three SNPs from coding regions were non-synonymous. Interestingly, the phenotype of No. 88 was still non-waxy, although seven nucleotides (AATTGGT) insertion at 2,993 bp led to 78 amino acids shorter. The rapid decline of r (2) in the sequenced region (exon 1-intron 1-exon 2) suggested a low level of linkage disequilibrium and limited haplotype structure. K (s) values and estimation of evolutionary events indicate early divergence of S. italica among cereal crops. This study suggested the Wx gene was one of the targets in the selection process during domestication.
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Affiliation(s)
- K Van
- Department of Plant Science, Seoul National University, San 56-1, Sillim-dong, Gwanak-gu, Seoul, 151-921, The Republic of Korea
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27
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Gonzalgo ML, Liang G. Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation. Nat Protoc 2007; 2:1931-6. [PMID: 17703204 DOI: 10.1038/nprot.2007.271] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.
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Affiliation(s)
- Mark L Gonzalgo
- Department of Urology, James Buchanan Brady Urological Institute, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287, USA.
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Zhang H, Mitrovski SM, Nuzzo RG. Microfluidic device for the discrimination of single-nucleotide polymorphisms in DNA oligomers using electrochemically actuated alkaline dehybridization. Anal Chem 2007; 79:9014-21. [PMID: 17973402 DOI: 10.1021/ac701660x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
This work describes an integrated microfluidic (mu-fl) device that can be used to effect separations that discriminate single-nucleotide polymorphisms (SNP) based on kinetic differences in the lability of perfectly matched (PM) and mismatched (MM) DNA duplexes during alkaline dehybridization. For this purpose a 21-base single-stranded DNA (ssDNA) probe sequence was immobilized on agarose-coated magnetic beads, that in turn can be localized within the channels of a poly(dimethylsiloxane) microfluidic device using an embedded magnetic separator. The PM and MM ssDNA targets were hybridized with the probe to form a mixture of PM and MM DNA duplexes using standard protocols, and the hydroxide ions necessary for mediating the dehybridization were generated electrochemically in situ by performing the oxygen reduction reaction (ORR) using O2 that passively permeates the device at a Pt working electrode (Pt-WE) embedded within the microfluidic channel system. The alkaline DNA dehybridization process was followed using fluorescence microscopy. The results of this study show that the two duplexes exhibit different kinetics of dehybridization, rate profiles that can be manipulated as a function of both the amount of the hydroxide ions generated and the mass-transfer characteristics of their transport within the device. This system is shown to function as a durable platform for effecting hybridization/dehybridization cycles using a nonthermal, electrochemical actuation mechanism, one that may enable new designs for lab-on-a-chip devices used in DNA analysis.
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Affiliation(s)
- Huaibin Zhang
- Department of Chemistry, University of Illinois at Urbana--Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA
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Cremonesi L, Ferrari M, Giordano PC, Harteveld CL, Kleanthous M, Papasavva T, Patrinos GP, Traeger-Synodinos J. An overview of current microarray-based human globin gene mutation detection methods. Hemoglobin 2007; 31:289-311. [PMID: 17654067 DOI: 10.1080/03630260701459366] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
The panoply of human globin gene mutation detection methods could become significantly enriched with the advent of microarray-based genotyping platforms. The aim of this article is to provide an overview of the current medium and high-throughput microarray-based globin gene mutation detection platforms, namely the microelectronic array, the "thalassochip" arrayed primer extension (APEX) technology and the single base extension methods. This article also outlines an emerging method based on multiple ligation probe amplification (MLPA) and discusses the implications of customized solutions for resequencing of genomic loci in relation to molecular genetic testing of hemoglobinopathies.
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Affiliation(s)
- Laura Cremonesi
- Genomic Unit for the Diagnosis of Human Pathologies, San Raffaele Scientific Institute, Milan, Italy
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30
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Zheng W, Wang Y, Luo J, Zhang F, Chen B, Lu Z. Microarray-based method to analyze methylation status of E-cadherin gene in leukemia. Clin Chim Acta 2007; 387:97-104. [PMID: 17964562 DOI: 10.1016/j.cca.2007.09.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2007] [Revised: 09/14/2007] [Accepted: 09/14/2007] [Indexed: 11/28/2022]
Abstract
BACKGROUND Aberrant DNA methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. Recently, several studies have indicated the aberrant methylation of E-cadherin gene could be a potential marker for leukemic patients. METHOD We used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated, but not methylated, cytosine into thymine within CpG islands of interest. The amplified product containing a pool of DNA fragments with altered nucleotide sequences was then hybridized with an oligonucleotide-based microarray. Five sets of oligonucleotide probes were designed to detect the methylation patterns of E-cadherin gene CpG islands in leukemia samples. The results were further validated by methylation-specific PCR (MSP). RESULTS We found that all leukemia samples were methylated at different levels within the target sequences. The specific regions (the CpG sites #16-19 and #20-22) were revealed as hotspots for methylation in leukemic patients. These results showed that the microarray assay could successfully detect methylation changes of E-cadherin gene in leukemia quantitatively. CONCLUSION The oligonucleotide-based microarray can be a quick and reliable tool to map methylation status in CpG islands. This established microarray could be potentially useful for clinical researches and diagnosis.
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Affiliation(s)
- Wenli Zheng
- State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
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Jeong S, Hahn Y, Rong Q, Pfeifer K. Accurate quantitation of allele-specific expression patterns by analysis of DNA melting. Genome Res 2007; 17:1093-100. [PMID: 17545578 PMCID: PMC1899120 DOI: 10.1101/gr.6028507] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
Epigenetic and genetic mechanisms can result in large differences in expression levels of the two alleles in a diploid organism. Furthermore, these differences may be critical to phenotypic variations among individuals. In this study, we present a novel procedure and algorithm to precisely and accurately quantitate the relative expression of each allele. This method uses the differential melting properties of DNAs differing at even a single base pair. By referring to the melting characteristics of the two pure alleles, the fractional contribution of the two alleles to any unknown mixture can be mathematically resolved. These methods are highly accurate and precise because each single melting reaction yields multiple data points for analysis. Finally, we discuss how this approach can be used more generally to accurately quantitate gene expression relative to known standards.
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Affiliation(s)
- Sangkyun Jeong
- Laboratory of Mammalian Genes and Development, National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892, USA
| | - Yoonsoo Hahn
- Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
| | - Qi Rong
- Laboratory of Mammalian Genes and Development, National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892, USA
| | - Karl Pfeifer
- Laboratory of Mammalian Genes and Development, National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892, USA
- Corresponding author.E-mail ; fax (301) 402-0543
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Abstract
PURPOSE OF REVIEW The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. RECENT FINDINGS New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. SUMMARY The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed 'haploChIP' allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.
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Affiliation(s)
- Sandra Pampín
- Department of Molecular Biology, Faculty of Medicine, University of Cantabria, Santander, Spain
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Bangsi D, Zhou J, Sun Y, Patel NP, Darga LL, Heilbrun LK, Powell IJ, Severson RK, Everson RB. Impact of a genetic variant in CYP3A4 on risk and clinical presentation of prostate cancer among white and African-American men. Urol Oncol 2006; 24:21-7. [PMID: 16414488 DOI: 10.1016/j.urolonc.2005.09.005] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2005] [Revised: 09/14/2005] [Accepted: 09/15/2005] [Indexed: 10/25/2022]
Abstract
Genes involved in androgen metabolism are strong candidates for having an important role in the pathogenesis of prostate cancer. CYP3A4, a protein in the cytochrome P-450 supergene family, facilitates the oxidative deactivation of testosterone. In previous studies, patients with the G variant of a genetic polymorphism in CYP3A4 had prostate cancers with clinically aggressive characteristics at diagnosis. The association was strongest among elderly men. We investigated whether the CYP3A4 variant was linked with the diagnosis or clinical presentation of prostate cancer in a case control study of a multiethnic urban population. Biologic specimens were genotyped for CYP3A4, and analyzed for the impact of this genotype on risk and tumor characteristics at presentation, controlling for the effect of several cofactors. The CYP3A4 variant was more common among African-Americans than among white men. Race-stratified analyses revealed little association between the CYP3A4 variant and prostate cancer risk among white men but were limited by the small number of white men with the CYP3A4 variant. Of African-American men, while the variant G allele was not associated with prostate cancer that had less aggressive characteristics, it was associated with risk of aggressive prostate cancer when men with the AG genotype (odds ratio = 9.3, 95% confidence interval 1.3-411) or GG genotype (odds ratio = 11.9 95% confidence interval 1.6-533) were compared with those with the AA genotype. The association between the CYP3A4 genotype and aggressive prostate cancer in African-American men is consistent with findings of other studies.
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Affiliation(s)
- Dieudonne Bangsi
- Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA
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Zhang D, Bai Y, Ge Q, Qiao Y, Wang Y, Chen Z, Lu Z. Microarray-based molecular margin methylation pattern analysis in colorectal carcinoma. Anal Biochem 2006; 355:117-24. [PMID: 16756932 DOI: 10.1016/j.ab.2006.04.048] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2006] [Revised: 04/13/2006] [Accepted: 04/25/2006] [Indexed: 11/18/2022]
Abstract
The positive surgical margins are associated with postsurgical recurrence in colorectal carcinoma patients, and molecular margin analysis is considered to be more sensitive in detecting preneoplastic lesions than is conventional histological margin examination. Here, we developed a microarray and established six calibration curves for hMLH1 gene methylation patterns analysis in 20 colorectal resected margin specimens and corresponding tumor tissue specimens as well as four normal tissue specimens. The results indicated that a moderate methylation level (8-42%) was found in 20 surgical margin tissues, extensive methylation (25-58%) was detected in 20 tumor tissues, and little or no methylation was observed in normal tissues. Of the six paired probes, the average methylation levels in 20 tumor tissues were 60, 35, 43, 53, 38, and 27%, whereas the average methylation levels of the six paired probes in 20 surgical margin tissues were 43, 16, 24, 28, 21, and 11%. Thus, this study demonstrated the feasibility of this assay for molecular assessment use. In addition, it will contribute significant information to our understanding of CpG island methylation for cancer diagnosis and postoperative recurrence.
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Affiliation(s)
- Dingdong Zhang
- State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, Jiangsu Province, China
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35
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Steemers FJ, Chang W, Lee G, Barker DL, Shen R, Gunderson KL. Whole-genome genotyping with the single-base extension assay. Nat Methods 2006; 3:31-3. [PMID: 16369550 DOI: 10.1038/nmeth842] [Citation(s) in RCA: 297] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2005] [Accepted: 11/10/2005] [Indexed: 02/07/2023]
Abstract
We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.
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Affiliation(s)
- Frank J Steemers
- Illumina, Inc., 9885 Towne Centre Drive, San Diego, California 92121, USA
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36
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Brena RM, Huang THM, Plass C. Quantitative assessment of DNA methylation: potential applications for disease diagnosis, classification, and prognosis in clinical settings. J Mol Med (Berl) 2006; 84:365-77. [PMID: 16416310 DOI: 10.1007/s00109-005-0034-0] [Citation(s) in RCA: 54] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2005] [Accepted: 11/29/2005] [Indexed: 12/31/2022]
Abstract
Deregulation of the epigenome is now recognized as a major mechanism involved in the development and progression of human diseases such as cancer. As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic modifications are reversible and leave the original DNA sequence intact. There is now evidence that the epigenetic landscape in humans undergoes modifications as the result of normal aging, with older individuals exhibiting higher levels of promoter hypermethylation compared to younger ones. Thus, it has been proposed that the higher incidence of certain disease in older individuals might be, in part, a consequence of an inherent change in the control and regulation of the epigenome. These observations are of remarkable clinical significance since the aberrant epigenetic changes characteristic of disease provide a unique platform for the development of new therapeutic approaches. In this review, we address the significance of DNA methylation changes that result or lead to disease, occur with aging, or may be the result of environmental exposure. We provide a detailed description of quantitative techniques currently available for the detection and analysis of DNA methylation and provide a comprehensive framework that may allow for the incorporation of protocols which include DNA methylation as a tool for disease diagnosis and classification, which could lead to the tailoring of therapeutic approaches designed to individual patient needs.
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Affiliation(s)
- Romulo Martin Brena
- Division of Human Cancer Genetics, Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA
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37
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Miyaki K, Sutani S, Kikuchi H, Takei I, Murata M, Watanabe K, Omae K. Increased risk of obesity resulting from the interaction between high energy intake and the Trp64Arg polymorphism of the beta3-adrenergic receptor gene in healthy Japanese men. J Epidemiol 2005; 15:203-10. [PMID: 16276029 PMCID: PMC7904380 DOI: 10.2188/jea.15.203] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
BACKGROUND: Few studies have investigated the interaction between the Trp64Arg polymorphism of the β3-adrenergic receptor gene (ADRB3) and environmental factors. This study aimed to investigate whether energy intake affects the relationship between this polymorphism and obesity. METHODS: Healthy Japanese men (n=295; age 46.1±11.5 years (mean ± standard deviation); waist circumference 83.9±9.3 cm; body mass index (BMI) 23.3±3.3 kg/m2) recruited in a Japanese chemical industry firm were eligible for analysis. Daily energy intake, protein, fat, and carbohydrate (PFC) ratio and daily physical activity were assessed by self-reported questionnaires. Genotyping for the polymorphism was performed with written informed consent. RESULTS: When the subjects were classified into two groups according to presence of the polymorphism, the groups were not significantly different in waist circumference or BMI. Quartile classification of energy intake, however, demonstrated a significantly larger ratio of obese subjects to non-obese subjects in the group with the polymorphism in the highest 4th quartile alone. Multiple logistic regression analysis also revealed that the presence of the polymorphism increased the risk of obesity significantly in the 4th quartile alone (adjusted odds ratio=3.37, 95% confidence interval=1.12-10.2). CONCLUSION: Presence of the polymorphism alone does not significantly increase the risk of obesity. However, high energy intake interacts with the polymorphism and leads to a significant increase in risk of obesity. The Trp64Arg polymorphism of ADRB3 warrants consideration, along with other polymorphisms involved in the development of obesity, for tailor-made prevention of obesity.
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Affiliation(s)
- Koichi Miyaki
- Department of Preventive Medicine and Public Health, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo, Japan.
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Van K, Hwang EY, Kim MY, Park HJ, Lee SH, Cregan PB. Discovery of SNPs in soybean genotypes frequently used as the parents of mapping populations in the United States and Korea. J Hered 2005; 96:529-35. [PMID: 15994422 DOI: 10.1093/jhered/esi069] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Single nucleotide polymorphisms (SNPs) including insertion/deletions (indels) serve as useful and informative genetic markers. The availability of high-throughput and inexpensive SNP typing systems has increased interest in the development of SNP markers. After fragments of genes were amplified with primers derived from 110 soybean GenBank ESTs, sequencing data of PCR products from 15 soybean genotypes from Korea and the United States were analyzed by SeqScape software to find SNPs. Among 35 gene fragments with at least one SNP among the 15 genotypes, SNPs occurred at a frequency of 1 per 2,038 bp in 16,302 bp of coding sequence and 1 per 191 bp in 16,960 bp of noncoding regions. This corresponds to a nucleotide diversity (theta) of 0.00017 and 0.00186, respectively. Of the 97 SNPs discovered, 78 or 80.4% were present in the six North American soybean mapping parents. The addition of "Hwaeomputkong," which originated from Japan, increased the number to 92, or 94.8% of the total number of SNPs present among the 15 genotypes. Thus, Hwaeomputkong and the six North American mapping parents provide a diverse set of soybean genotypes that can be successfully used for SNP discovery in coding DNA and closely associated introns and untranslated regions.
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Affiliation(s)
- K Van
- Department of Plant Science, Seoul National University, Seoul 151-921, Republic of Korea
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39
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Ge C, Miao W, Ji M, Gu N. Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of p16 INK4A gene. Anal Bioanal Chem 2005; 383:651-9. [PMID: 16132144 DOI: 10.1007/s00216-005-0032-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2005] [Revised: 07/11/2005] [Accepted: 07/14/2005] [Indexed: 10/25/2022]
Abstract
A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ( INK4A ) gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ( INK4A ) gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.
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Affiliation(s)
- Cunwang Ge
- School of Chemistry and Chemical Engineering, Nantong University, Nantong 226007, People's Republic of China.
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40
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Doi Y, Yamamoto Y, Inagaki S, Shigeta Y, Miyaishi S, Ishizu H. A new method for ABO genotyping using a multiplex single-base primer extension reaction and its application to forensic casework samples. Leg Med (Tokyo) 2005; 6:213-23. [PMID: 15363446 DOI: 10.1016/j.legalmed.2004.05.005] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2004] [Revised: 04/23/2004] [Accepted: 05/18/2004] [Indexed: 10/26/2022]
Abstract
We developed a new method for ABO genotyping using a multiplex single-base primer extension reaction. The method allows for the simultaneous detection of six SNP sites in the ABO gene (nt 261, 297, 681, 703, 802, and 803) and the determination of ABO genotypes from their combinations. It enabled ABO genotyping of all samples of peripheral blood DNA extracted from 103 Japanese individuals, and had a highly satisfactory detection sensitivity being capable of genotyping 0.1 ng of genomic DNA. Using this method, we were able to determine ABO genotypes of minute stain samples, heated bloodstains, aged bloodstains and mixed samples. Experiments with samples from 26 animal species and bacterial samples to test the species-specificity of the method showed that genotyping was possible in the chimpanzee and gorilla, but their genotypes were extremely rare in humans. In addition, we applied this method to casework samples, and successfully determined ABO genotypes of bones, teeth, muscles, organs, nails, and semen-contaminated vaginal fluid in which ABO grouping by conventional serological techniques was not possible. This new method enables the sensitive, simultaneous detection of six SNP sites in the ABO gene by two specific reactions, i.e. PCR and a primer extension reaction. Therefore, it holds promise as an effective method of ABO genotyping particularly for forensic samples.
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Affiliation(s)
- Yusuke Doi
- Department of Legal Medicine, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-Cho, Okayama City 700-8558, Japan.
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41
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Hou P, Ji M, Li S, He N, Lu Z. High-throughput method for detecting DNA methylation. ACTA ACUST UNITED AC 2005; 60:139-50. [PMID: 15262448 DOI: 10.1016/j.jbbm.2004.05.001] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2004] [Revised: 05/04/2004] [Accepted: 05/05/2004] [Indexed: 11/20/2022]
Abstract
Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related gene may be useful for cancer diagnosis or the detection of recurrence. However, most of the studies have focused on a single gene only and gave little information about the concurrent methylation status of multiple genes. In this study, we attempted to develop a microarray method coupled with linker-PCR for detecting methylation status of multiple genes in the tumor tissue. A series of synthesized oligonucleotides were synthesised and purified to completely match with 16 investigated targets. Then they were immobilized on the aldehyde-coated glass slide to fabricate a DNA microarray for detecting methylation status of these genes. The results indicated that these genes were all methylated in the positive control. However, no methylated was found in these genes for the negative control. Only p16 and p15 genes were methylated in investigated genes for the gastric tumor tissue, whereas others were not methylated. The above results were validated by bisulfite DNA sequencing. Our experiments successfully demonstrated that the DNA microarray could be applied as a high-throughput tool to determine methylation status of the investigated genes.
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Affiliation(s)
- Peng Hou
- Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
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42
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Sobrino B, Brión M, Carracedo A. SNPs in forensic genetics: a review on SNP typing methodologies. Forensic Sci Int 2005; 154:181-94. [PMID: 16182964 DOI: 10.1016/j.forsciint.2004.10.020] [Citation(s) in RCA: 229] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2004] [Revised: 10/10/2004] [Accepted: 10/15/2004] [Indexed: 11/22/2022]
Abstract
There is an increasing interest in single nucleotide polymorphism (SNP) typing in the forensic field, not only for the usefulness of SNPs for defining Y chromosome or mtDNA haplogroups or for analyzing the geographical origin of samples, but also for the potential applications of autosomal SNPs. The interest of forensic researchers in autosomal SNPs has been attracted due to the potential advantages in paternity testing because of the low mutation rates and specially in the analysis of degraded samples by use of short amplicons. New SNP genotyping methods, chemistries and platforms are continuously being developed and it is often difficult to be keeping up to date and to decide on the best technology options available. This review offers to the reader a state of the art of SNP genotyping technologies with the advantages and disadvantages of the different chemistries and platforms for different forensic requirements.
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Affiliation(s)
- Beatriz Sobrino
- Institute of Legal Medicine, University of Santiago de Compostela, San Francisco s/n, Spain.
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43
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Hou P, Shen JY, Ji MJ, He NY, Lu ZH. Microarray-based method for detecting methylation changes of p16 Ink4a gene 5’-CpG islands in gastric carcinomas. World J Gastroenterol 2004; 10:3553-8. [PMID: 15534905 PMCID: PMC4611991 DOI: 10.3748/wjg.v10.i24.3553] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of high-throughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer.
METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP).
RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover, it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP.
CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.
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Affiliation(s)
- Peng Hou
- Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, Jiangsu Province, China
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Vaccari G, Conte M, Morelli L, Di Guardo G, Petraroli R, Agrimi U. Primer extension assay for prion protein genotype determination in sheep. Mol Cell Probes 2004; 18:33-7. [PMID: 15036367 DOI: 10.1016/j.mcp.2003.06.001] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2003] [Accepted: 06/26/2003] [Indexed: 11/19/2022]
Abstract
Scrapie is a transmissible spongiform encephalopathy (TSE) which affects sheep and goats. TSEs are characterised by the conversion of the cellular prion protein (PrP(C)) into the pathological form PrP(Sc). The occurrence of scrapie in sheep is influenced by polymorphisms in the PrP gene; in particular, three codons (136, 154 and 171) are important in conditioning the susceptibility/resistance of sheep to the disease, with the Val/Val(136) Arg/Arg(154) Gln/Gln(171) genotype being the most susceptible and the Ala/Ala(136) Arg/Arg(154) Arg/Arg(171), the most resistant one. The latter genotype seems to confer, in sheep, resistance to the oral infection with bovine spongiform encephalopathy, as well. The selection of genetically resistant sheep populations represents the basis of the recent strategies against ovine TSE in the European Union (EU). Herein, we describe a rapid and simple method, based on the primer extension technique, for PrP genotype determination at codons 136, 154 and 171. Intra-laboratory validation of the method showed accuracy levels comparable to those of sequencing analysis. Such method could be used for both the application of the EU policies requiring PrP genotype analysis in all ovine TSE cases, and the large-scale genotyping claimed by the implementation of breeding programmes for genetic resistance to TSE in sheep.
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Affiliation(s)
- G Vaccari
- Laboratory of Veterinary Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome 00161, Italy.
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Powell IJ, Zhou J, Sun Y, Sakr WA, Patel NP, Heilbrun LK, Everson RB. CYP3A4 GENETIC VARIANT AND DISEASE-FREE SURVIVAL AMONG WHITE AND BLACK MEN AFTER RADICAL PROSTATECTOMY. J Urol 2004; 172:1848-52. [PMID: 15540736 DOI: 10.1097/01.ju.0000142779.76603.be] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
PURPOSE Prostate cancer is an androgen sensitive disease. Cytochrome P450 3A4 (CYP3A4) oxidatively deactivates testosterone by converting it to biologically less active metabolites. Previous studies suggest that a germline genetic variant in the 5' regulatory region of the gene may interfere with deactivation and increase the risk of clinically advanced prostate cancer. We investigated the impact of this polymorphism on the risk of recurrence after prostatectomy. MATERIALS AND METHODS We assembled clinical data and analyzed specimens from a large series of patients who underwent prostatectomy who were carefully staged at a single institution and had 5 to 10 years of prospective clinical followup. The series included 428 white men and 309 black men. RESULTS Stage, Gleason score or preoperative prostate specific antigen strongly predicted progression-free survival (PFS) but were not associated with CYP3A4 genotypes. There was a strong association between race and genotype (p = 0.00002) in that 8% of white men and 83% of black men had 1 or more copies of the G allele. When both races were included genotype was associated with PFS (hazard ratio [HR] 1.27, CI 1.08-1.27, p = 0.005). In race specific analyses increasing copies of the G allele were associated with poorer PFS among white men (HR 1.98, CI 1.06-3.70, p = 0.03) but had little impact on PFS among black men (HR 1.004, CI 0.77-1.32, p = 0.97). CONCLUSIONS The CYP3A4 genotype studied was not associated with pathological features of prostate cancer for men of either race. Unstratified analyses of men of both races and stratified analyses of white men demonstrated poorer PFS after prostatectomy for those with the G allele, but the G allele did not predict PFS among black men.
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Affiliation(s)
- Isaac J Powell
- Barbara Ann Karmanos Cancer Institute, Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
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Mo QH, Zhu H, Li LY, Xu XM. Reliable and High-Throughput Mutation Screening for β-Thalassemia by a Single-Base Extension/Fluorescence Polarization Assay. ACTA ACUST UNITED AC 2004; 8:257-62. [PMID: 15727248 DOI: 10.1089/gte.2004.8.257] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.
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Affiliation(s)
- Qiu-Hua Mo
- Department of Medical Genetics, First Military Medical University, Guangzhou 510515, Guangdong Province, PR China
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van Moorsel CHM, van Wijngaarden EE, Fokkema IFAC, den Dunnen JT, Roos D, van Zwieten R, Giordano PC, Harteveld CL. β-Globin mutation detection by tagged single-base extension and hybridization to universal glass and flow-through microarrays. Eur J Hum Genet 2004; 12:567-73. [PMID: 15069457 DOI: 10.1038/sj.ejhg.5201192] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
To test the feasibility of developing a diagnostic microarray for a specific disease, we selected all pathogenic changes of the beta-globin gene occurring at a frequency >/=1% in the multi-ethnic Dutch population for analysis. A tagged single-base extension (SBE) approach was used to detect 19 different mutations causing beta-thalassemia or abnormal hemoglobins. In the SBE reaction, the primers were elongated at the 3'site with a fluorescently labeled dideoxyribonucleotide triphosphate (ddNTP) complementary to the mutation, following tag hybridization to a glass or flow-through microarray. We compared the performance of a generic glass array and a porous system, by testing each mutation separately using heterozygous carriers and by screening a cohort of 40 unknown beta-thalassemia carriers and patients. The results were verified by direct sequencing. The microarray system was able to detect 17 beta-globin mutations simultaneously with >95% accuracy in a single SBE reaction. The flow-through array performed slightly better (96%), but the main advantages of the system included real-time data recording and a considerable time saving achieved through a reduced hybridization time.
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Affiliation(s)
- Coline H M van Moorsel
- Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
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Ji M, Hou P, Li S, He N, Lu Z. Colorimetric silver detection of methylation using DNA microarray coupled with linker-PCR. Clin Chim Acta 2004; 342:145-53. [PMID: 15026275 DOI: 10.1016/j.cccn.2003.12.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2003] [Revised: 12/15/2003] [Accepted: 12/15/2003] [Indexed: 10/26/2022]
Abstract
BACKGROUND Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Detection of promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or the detection of recurrence. Recently, several DNA microarray methods have been developed to detect the methylation status of the multiple genes. However, the microarrays are currently detected in fluorescence using a sophisticated laser-based scanner. These methods are limited by their lower sensitivity. METHODS We present a sensitive colorimetric silver method coupled with linker-PCR to detect methylation status of four genes, including p16, E-cadherin, VHL, and hMLH1. The signal generated with this method results from the precipitation of silver onto nanogold particles bound to streptavidin used to detect biotinylated DNA. RESULTS We show that p16, E-cadherin, VHL, and hMLH1 were all methylated in the positive control. However, no methylation was found in these genes for the negative control. P16 gene was only methylated in the sample, whereas others were not methylated. Furthermore, as little as 0.1 fmol of target DNA amplicons could be detected on the arrays. The results were further validated by methylation-specific PCR (MSP) and bisulfite DNA sequencing. CONCLUSIONS The colorimetric silver detection of DNA microarray could be used as an inexpensive, useful, and sensitive tool to detect methylation status of the multiple genes in the future.
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Affiliation(s)
- Meiju Ji
- Chien-Shiung Wu Laboratory, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China
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Ren B, Zhou JM, Komiyama M. Straightforward detection of SNPs in double-stranded DNA by using exonuclease III/nuclease S1/PNA system. Nucleic Acids Res 2004; 32:e42. [PMID: 14982961 PMCID: PMC390314 DOI: 10.1093/nar/gnh039] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Single-nucleotide polymorphisms (SNPs) in double-stranded DNA (dsDNA) have been straightforwardly genotyped by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Peptide nucleic acid (PNA), a DNA analog, was used as a probe molecule. In its presence, genomic dsDNA was first treated with exonuclease III and then with nuclease S1. By these one-pot reactions, single-stranded DNA fragments including the SNP sites were formed in situ. These fragments were directly analyzed by MALDI-TOF MS, and the identity of the DNA base at the SNP site was determined in terms of mass number. By using two or more PNA probes simultaneously, multiplex analysis was also successful. Various genotypes of apolipoprotein E gene (epsilon2/epsilon2, epsilon3/epsilon3, epsilon4/epsilon4, epsilon2/epsilon3 and epsilon3/epsilon4) were identified from dsDNA obtained by PCR from corresponding patients.
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Affiliation(s)
- Binzhi Ren
- Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan
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Törjék O, Berger D, Meyer RC, Müssig C, Schmid KJ, Rosleff Sörensen T, Weisshaar B, Mitchell-Olds T, Altmann T. Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2003; 36:122-140. [PMID: 12974817 DOI: 10.1046/j.1365-313x.2003.01861.x] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/24/2023]
Abstract
The major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)-based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col-0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single-tube/single-capillary format. In addition, for 110 framework markers matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col-0 and C24. Subsets of 10-62 of the established markers discriminate between various combinations of the accessions Col-0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col-0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) x Col-0 cross.
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Affiliation(s)
- O Törjék
- University of Potsdam, Institute of Biochemistry and Biology-Genetics, Golm, Germany
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