The Experts below are selected from a list of 738 Experts worldwide ranked by ideXlab platform
Lloyd M Smith - One of the best experts on this subject based on the ideXlab platform.
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analysis of single nucleotide polymorphisms by primer extension and matrix assisted laser desorption ionization time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2000Co-Authors: Zhengdong Fei, Lloyd M SmithAbstract:A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence-tagged sites (STSs). Copyright © 2000 John Wiley & Sons, Ltd.
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MALDI- TOF mass spectrometric typing of single nucleotide polymorphisms with mass-tagged ddNTPs. Nucleic Acids Res 26:2827–2828
1998Co-Authors: Zhengdong Fei, Tetsuyoshi Ono, Lloyd M SmithAbstract:A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based method has recently been reported for the typing of single nucleotide polymorphisms using single nucleotide primer extension. This method is limited in some cases by the resolution of the mass determination, as the mass difference between nucleotides can be as little as 9 Da (the difference between A and T). A variation of this method is described here in which a mass-tagged Dideoxynucleotide is employed in the primer extension reactions in place of the unmodified Dideoxynucleotide. The increased mass difference due to the presence of the mass-tags substantially improves the accuracy and versatility of the procedure
Zhengdong Fei - One of the best experts on this subject based on the ideXlab platform.
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analysis of single nucleotide polymorphisms by primer extension and matrix assisted laser desorption ionization time of flight mass spectrometry
Rapid Communications in Mass Spectrometry, 2000Co-Authors: Zhengdong Fei, Lloyd M SmithAbstract:A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence-tagged sites (STSs). Copyright © 2000 John Wiley & Sons, Ltd.
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MALDI- TOF mass spectrometric typing of single nucleotide polymorphisms with mass-tagged ddNTPs. Nucleic Acids Res 26:2827–2828
1998Co-Authors: Zhengdong Fei, Tetsuyoshi Ono, Lloyd M SmithAbstract:A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry based method has recently been reported for the typing of single nucleotide polymorphisms using single nucleotide primer extension. This method is limited in some cases by the resolution of the mass determination, as the mass difference between nucleotides can be as little as 9 Da (the difference between A and T). A variation of this method is described here in which a mass-tagged Dideoxynucleotide is employed in the primer extension reactions in place of the unmodified Dideoxynucleotide. The increased mass difference due to the presence of the mass-tags substantially improves the accuracy and versatility of the procedure
C Richardson - One of the best experts on this subject based on the ideXlab platform.
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a single residue in dna polymerases of the escherichia coli dna polymerase i family is critical for distinguishing between deoxy and dideoxyribonucleotides
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Stanley Tabor, C RichardsonAbstract:Abstract Bacteriophage T7 DNA polymerase efficiently incorporates a chain-terminating Dideoxynucleotide into DNA, in contrast to the DNA polymerases from Escherichia coli and Thermus aquaticus. The molecular basis for this difference has been determined by constructing active site hybrids of these polymerases. A single hydroxyl group on the polypeptide chain is critical for selectivity. Replacing tyrosine-526 of T7 DNA polymerase with phenylalanine increases discrimination against the four Dideoxynucleotides by > 2000-fold, while replacing the phenylalanine at the homologous position in E. coli DNA polymerase I (position 762) or T. aquaticus DNA polymerase (position 667) with tyrosine decreases discrimination against the four Dideoxynucleotides 250- to 8000-fold. These mutations allow the engineering of new DNA polymerases with enhanced properties for use in DNA sequence analysis.
Eric A Schon - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial single nucleotide polymorphism genotyping by matrix assisted laser desorption ionization time of flight mass spectrometry using cleavable biotinylated Dideoxynucleotides
Analytical Biochemistry, 2012Co-Authors: Chunmei Qiu, Shiv Kumar, Jia Guo, Shundi Shi, Sergey Kalachikov, James J Russo, Ali Naini, Eric A SchonAbstract:Characterization of mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) and mutations is crucial for disease diagnosis, which requires accurate and sensitive detection methods and quantification due to mitochondrial heteroplasmy. We report here the characterization of mutations for myoclonic epilepsy with ragged red fibers syndrome using chemically cleavable biotinylated Dideoxynucleotides and a mass spectrometry (MS)-based solid phase capture (SPC) single base extension (SBE) assay. The method effectively eliminates unextended primers and primer dimers, and the presence of cleavable linkers between the base and biotin allows efficient desalting and release of the DNA products from solid phase for MS analysis. This approach is capable of high multiplexing, and the use of different length linkers for each of the purines and each of the pyrimidines permits better discrimination of the four bases by MS. Both homoplasmic and heteroplasmic genotypes were accurately determined on different mtDNA samples. The specificity of the method for mtDNA detection was validated by using mitochondrial DNA-negative cells. The sensitivity of the approach permitted detection of less than 5% mtDNA heteroplasmy levels. This indicates that the SPC–SBE approach based on chemically cleavable biotinylated Dideoxynucleotides and MS enables rapid, accurate, and sensitive genotyping of mtDNA and has broad applications for genetic analysis.
Diana Tabbaa - One of the best experts on this subject based on the ideXlab platform.
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UNIT 2.12SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform
2015Co-Authors: Stacey Gabriel, Liuda Ziaugra, Diana TabbaaAbstract:The method for SNP genotyping described in this unit is based on the commercially available Sequenom MassARRAY platform. The assay consists of an initial locus-specific PCR reaction, followed by single base extension using mass-modified Dideoxynucleotide terminators of an oligonucleotide primer which anneals immediately upstream of the polymorphic site of interest. Using MALDI-TOF mass spectrometry, the distinct mas
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SNP genotyping using the Sequenom MassARRAY iPLEX platform.
Current protocols in human genetics, 2009Co-Authors: Stacey Gabriel, Liuda Ziaugra, Diana TabbaaAbstract:The method for SNP genotyping described in this unit is based on the commercially available Sequenom MassARRAY platform. The assay consists of an initial locus-specific PCR reaction, followed by single base extension using mass-modified Dideoxynucleotide terminators of an oligonucleotide primer which anneals immediately upstream of the polymorphic site of interest. Using MALDI-TOF mass spectrometry, the distinct mass of the extended primer identifies the SNP allele.