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Axel Brennicke - One of the best experts on this subject based on the ideXlab platform.
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Using multiplex Single-Base Extension typing to screen for mutants defective in RNA editing
Nature Protocols, 2012Co-Authors: Mizuki Takenaka, Axel BrennickeAbstract:RNA editing is an RNA maturation process that changes the nucleotide present at particular positions (editing sites) in specific RNAs; in plant organelles, the most common nucleotide change is from cytidine (C) to uridine (U). In a mutant suspected of affecting RNA editing, all known editing sites have to be analyzed. Therefore, to screen a population of mutants, all individuals must be analyzed at every editing site. We describe a multiplex single-nucleotide polymorphism (SNP)–typing procedure to economically screen a mutant individual or population for differences at hundreds of nucleotide positions in RNA or DNA. By using this protocol, we have previously identified mutants defective in RNA editing in a randomly mutated population of Arabidopsis thaliana. The procedure requires 2–3 weeks to identify the individual plant in the mutant population. The time required to locate the mutated gene is between 3 and 24 months in Arabidopsis . Although this procedure has been developed to study RNA editing in plants, it could also be used to investigate other RNA modification processes. It could also be adapted to investigate RNA editing in other organisms.
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Using multiplex Single-Base Extension typing to screen for mutants defective in RNA editing.
Nature protocols, 2012Co-Authors: Mizuki Takenaka, Axel BrennickeAbstract:Using multiplex Single-Base Extension typing to screen for mutants defective in RNA editing
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Multiplex Single-Base Extension typing to identify nuclear genes required for RNA editing in plant organelles
Nucleic acids research, 2008Co-Authors: Mizuki Takenaka, Axel BrennickeAbstract:We developed a multiplex Single-Base Extension single-nucleotide polymorphism-typing procedure for screening large numbers of plants for mutations in mitochondrial RNA editing. The high sensitivity of the approach detects changes in the RNA editing status generated in total cellular cDNA from pooled RNA preparations of up to 50 green plants. The method has been employed to tag several nuclear encoded genes required for RNA editing at specific sites in mitochondria of Arabidopsis thaliana. This approach will allow large-scale screening for mutations in genes encoding trans-factors for many types of RNA editing as well as for other RNA modifications.
Niels Morling - One of the best experts on this subject based on the ideXlab platform.
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Forensic SNP genotyping with SNaPshot: Technical considerations for the development and optimization of multiplexed SNP assays.
Forensic science review, 2017Co-Authors: M. Fondevila, Angel Carracedo, Claus Børsting, Niels Morling, Christopher Phillips, De La Puente M, Maria Victoria LareuAbstract:This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot Single-Base Extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides an advantage. In order to provide the basis for developing such expertise, we cover in this paper the most challenging aspects of the SNaPshot technology, focusing on the steps taken to design primer sets, optimize the PCR and Single-Base Extension chemistries, and the important features of the peak patterns observed in typical forensic SNP profiles using SNaPshot. With that purpose in mind, we provide guidelines and troubleshooting for multiplex-SNaPshot-oriented primer design and the resulting capillary electrophoresis (CE) profile interpretation (covering the most commonly observed artifacts and expected departures from the ideal conditions).
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Typing of 49 autosomal SNPs by single base Extension and capillary electrophoresis for forensic genetic testing.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Claus Børsting, Carmen Tomas, Niels MorlingAbstract:We describe a method for simultaneous amplification of 49 autosomal single nucleotide polymorphisms (SNPs) by multiplex PCR and detection of the SNP alleles by single base Extension (SBE) and capillary electrophoresis. All the SNPs may be amplified from only 100 pg of genomic DNA and the length of the amplicons range from 65 to 115 bp. The high sensitivity and the short amplicon sizes make the assay very suitable for typing of degraded DNA samples, and the low mutation rate of SNPs makes the assay very useful for relationship testing. Combined, these advantages make the assay well suited for disaster victim identifications, where the DNA from the victims may be highly degraded and the victims are identified via investigation of their relatives. The assay was validated according to the ISO 17025 standard and used for routine case work in our laboratory.
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Determination of cis/trans phase of variations in the MC1R gene with allele-specific PCR and single base Extension.
Electrophoresis, 2008Co-Authors: Jonas Mengel-from, Claus Børsting, Juan J. Sanchez, Hans Eiberg, Niels MorlingAbstract:The MC1R gene encodes a protein with key regulatory functions in the melanin synthesis. A multiplex PCR and a multiplex single base Extension protocol were established for genotyping six exonic MC1R variations highly penetrant for red hair (R), four exonic MC1R variations weakly penetrant for red hair (r), two frameshift variations highly penetrant for red hair (R) and three variations in the promoter region. We genotyped 600 individuals from Denmark using either CE or MALDI-TOF MS as the detection platform. A total of 62 individuals were genotyped R/R and among the 62 individuals, 57 had red hair and five had blond hair colour. Two different R alleles may be located in cis (RR/-) position or trans (R/R) position, and the phenotype associated with RR/- and R/R may be different. Two allele-specific PCRs were established with primers targeting the -G445A variation in the MC1R promoter and the allele-specific PCR products were used in the multiplex single base Extension assay. In all 62 individuals, the MC1R variants were situated in trans position. Another 18 individuals with red hair colour were either genotyped R/- or R/r, suggesting that other genes influence hair colour.
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Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric Single-Base Extension primers and detection by MALDI-TOF mass spectrometry.
Analytical chemistry, 2005Co-Authors: J. Mengel-jørgensen, Claus Børsting, Juan J. Sanchez, F Kirpekar, Niels MorlingAbstract:A novel Single-Base Extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3‘-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000−5500 m/z range, ...
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typing of multiple single nucleotide polymorphisms using ribonuclease cleavage of dna rna chimeric single base Extension primers and detection by maldi tof mass spectrometry
Analytical Chemistry, 2005Co-Authors: J Mengeljorgensen, Claus Børsting, Juan J. Sanchez, F Kirpekar, Niels MorlingAbstract:A novel Single-Base Extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3‘-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000−5500 m/z range, ...
Mizuki Takenaka - One of the best experts on this subject based on the ideXlab platform.
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Using multiplex Single-Base Extension typing to screen for mutants defective in RNA editing
Nature Protocols, 2012Co-Authors: Mizuki Takenaka, Axel BrennickeAbstract:RNA editing is an RNA maturation process that changes the nucleotide present at particular positions (editing sites) in specific RNAs; in plant organelles, the most common nucleotide change is from cytidine (C) to uridine (U). In a mutant suspected of affecting RNA editing, all known editing sites have to be analyzed. Therefore, to screen a population of mutants, all individuals must be analyzed at every editing site. We describe a multiplex single-nucleotide polymorphism (SNP)–typing procedure to economically screen a mutant individual or population for differences at hundreds of nucleotide positions in RNA or DNA. By using this protocol, we have previously identified mutants defective in RNA editing in a randomly mutated population of Arabidopsis thaliana. The procedure requires 2–3 weeks to identify the individual plant in the mutant population. The time required to locate the mutated gene is between 3 and 24 months in Arabidopsis . Although this procedure has been developed to study RNA editing in plants, it could also be used to investigate other RNA modification processes. It could also be adapted to investigate RNA editing in other organisms.
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Using multiplex Single-Base Extension typing to screen for mutants defective in RNA editing.
Nature protocols, 2012Co-Authors: Mizuki Takenaka, Axel BrennickeAbstract:Using multiplex Single-Base Extension typing to screen for mutants defective in RNA editing
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Identifying specific trans-factors of RNA editing in plant mitochondria by multiplex single base Extension typing.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Mizuki TakenakaAbstract:The multiplex single base Extension SNP-typing procedure outlined here can be employed to screen large numbers of plants for mutations in nuclear genes that affect mitochondrial RNA editing. The high -sensitivity of this method allows high-throughput analysis of individual plants altered in RNA editing at given sites in total cellular cDNA from pooled RNA preparations of up to 50 green plants. The method can be used for large-scale screening for mutations in genes encoding trans-factors for specific RNA -editing sites. Several nuclear encoded genes involved in RNA editing at specific sites in mitochondria of Arabidopsis thaliana have been identified by this approach.
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Multiplex Single-Base Extension typing to identify nuclear genes required for RNA editing in plant organelles
Nucleic acids research, 2008Co-Authors: Mizuki Takenaka, Axel BrennickeAbstract:We developed a multiplex Single-Base Extension single-nucleotide polymorphism-typing procedure for screening large numbers of plants for mutations in mitochondrial RNA editing. The high sensitivity of the approach detects changes in the RNA editing status generated in total cellular cDNA from pooled RNA preparations of up to 50 green plants. The method has been employed to tag several nuclear encoded genes required for RNA editing at specific sites in mitochondria of Arabidopsis thaliana. This approach will allow large-scale screening for mutations in genes encoding trans-factors for many types of RNA editing as well as for other RNA modifications.
Claus Børsting - One of the best experts on this subject based on the ideXlab platform.
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Forensic SNP genotyping with SNaPshot: Technical considerations for the development and optimization of multiplexed SNP assays.
Forensic science review, 2017Co-Authors: M. Fondevila, Angel Carracedo, Claus Børsting, Niels Morling, Christopher Phillips, De La Puente M, Maria Victoria LareuAbstract:This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot Single-Base Extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides an advantage. In order to provide the basis for developing such expertise, we cover in this paper the most challenging aspects of the SNaPshot technology, focusing on the steps taken to design primer sets, optimize the PCR and Single-Base Extension chemistries, and the important features of the peak patterns observed in typical forensic SNP profiles using SNaPshot. With that purpose in mind, we provide guidelines and troubleshooting for multiplex-SNaPshot-oriented primer design and the resulting capillary electrophoresis (CE) profile interpretation (covering the most commonly observed artifacts and expected departures from the ideal conditions).
-
Typing of 49 autosomal SNPs by single base Extension and capillary electrophoresis for forensic genetic testing.
Methods in molecular biology (Clifton N.J.), 2011Co-Authors: Claus Børsting, Carmen Tomas, Niels MorlingAbstract:We describe a method for simultaneous amplification of 49 autosomal single nucleotide polymorphisms (SNPs) by multiplex PCR and detection of the SNP alleles by single base Extension (SBE) and capillary electrophoresis. All the SNPs may be amplified from only 100 pg of genomic DNA and the length of the amplicons range from 65 to 115 bp. The high sensitivity and the short amplicon sizes make the assay very suitable for typing of degraded DNA samples, and the low mutation rate of SNPs makes the assay very useful for relationship testing. Combined, these advantages make the assay well suited for disaster victim identifications, where the DNA from the victims may be highly degraded and the victims are identified via investigation of their relatives. The assay was validated according to the ISO 17025 standard and used for routine case work in our laboratory.
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Determination of cis/trans phase of variations in the MC1R gene with allele-specific PCR and single base Extension.
Electrophoresis, 2008Co-Authors: Jonas Mengel-from, Claus Børsting, Juan J. Sanchez, Hans Eiberg, Niels MorlingAbstract:The MC1R gene encodes a protein with key regulatory functions in the melanin synthesis. A multiplex PCR and a multiplex single base Extension protocol were established for genotyping six exonic MC1R variations highly penetrant for red hair (R), four exonic MC1R variations weakly penetrant for red hair (r), two frameshift variations highly penetrant for red hair (R) and three variations in the promoter region. We genotyped 600 individuals from Denmark using either CE or MALDI-TOF MS as the detection platform. A total of 62 individuals were genotyped R/R and among the 62 individuals, 57 had red hair and five had blond hair colour. Two different R alleles may be located in cis (RR/-) position or trans (R/R) position, and the phenotype associated with RR/- and R/R may be different. Two allele-specific PCRs were established with primers targeting the -G445A variation in the MC1R promoter and the allele-specific PCR products were used in the multiplex single base Extension assay. In all 62 individuals, the MC1R variants were situated in trans position. Another 18 individuals with red hair colour were either genotyped R/- or R/r, suggesting that other genes influence hair colour.
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Analysis of 29 Y-chromosome SNPs in a single multiplex useful to predict the geographic origin of male lineages
International Congress Series, 2006Co-Authors: Maria Brion, Claus Børsting, Juan J. Sanchez, Kinga Balogh, C.r. Thacker, Alejandro Blanco-verea, Beate Stradmann-bellinghausen, Magdalena Bogus, Denise Syndercombe-court, Peter M. SchneiderAbstract:Abstract The European Consortium “High throughput analysis of single nucleotide polymorphisms for the forensic identification of persons—SNPforID” has performed a selection of candidate Y-chromosome SNPs (single nucleotide polymorphisms) for making inferences on the geographic origin of an unknown sample. A “Major Y chromosome haplogroup typing kit” has been developed, which allows the multiplex amplification of 29 SNPs in a single reaction followed by a single base Extension (SBE) reaction (minisequencing) and separation of the resulting Extension products by capillary electrophoresis.
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Typing of multiple single-nucleotide polymorphisms using ribonuclease cleavage of DNA/RNA chimeric Single-Base Extension primers and detection by MALDI-TOF mass spectrometry.
Analytical chemistry, 2005Co-Authors: J. Mengel-jørgensen, Claus Børsting, Juan J. Sanchez, F Kirpekar, Niels MorlingAbstract:A novel Single-Base Extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled ddNTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3‘-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000−5500 m/z range, ...
Chiacheng Hung - One of the best experts on this subject based on the ideXlab platform.
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Quantification of relative gene dosage by Single-Base Extension and high-performance liquid chromatography: application to the SMN1/SMN2 gene.
Analytical chemistry, 2005Co-Authors: Chiacheng Hung, Chihping Chen, Yuhjyh Jong, Chian Chen, Wenfang Cheng, Chien-nan Lee, Win-li LinAbstract:One of the most commonly used techniques for genotyping of single-nucleotide polymorphism (SNP) is detection of Single-Base Extensions (SBEs). We present a new, rapid, simple, and highly reliable method for accurate quantification of SNP variants in a single reaction. Our approach is based on SBE detection coupled with high-performance liquid chromatography (HPLC) quantification. To demonstrate the utility of our approach, we report data to determine the gene dosage for relative amounts of alleles in a homologous gene, allowing detection of mutation causing exon skipping in human SMN genes to determine the ratio between the copy numbers of the SMN1/SMN2 gene. We successfully determined the relative ratio of the SMN1 and SMN2 genes and showed assay characteristics using the SBE reaction coupled with HPLC. This assay approach readily scaled to high parallelization with multiplex SBE reactions in a single sample screened in one analysis. By screening for particular SNP genotypes, this assay can be used to de...
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quantification of relative gene dosage by single base Extension and high performance liquid chromatography application to the smn1 smn2 gene
Analytical Chemistry, 2005Co-Authors: Chiacheng Hung, Chihping Chen, Yuhjyh Jong, Chian Chen, Wenfang Cheng, Yining SuAbstract:One of the most commonly used techniques for genotyping of single-nucleotide polymorphism (SNP) is detection of Single-Base Extensions (SBEs). We present a new, rapid, simple, and highly reliable method for accurate quantification of SNP variants in a single reaction. Our approach is based on SBE detection coupled with high-performance liquid chromatography (HPLC) quantification. To demonstrate the utility of our approach, we report data to determine the gene dosage for relative amounts of alleles in a homologous gene, allowing detection of mutation causing exon skipping in human SMN genes to determine the ratio between the copy numbers of the SMN1/SMN2 gene. We successfully determined the relative ratio of the SMN1 and SMN2 genes and showed assay characteristics using the SBE reaction coupled with HPLC. This assay approach readily scaled to high parallelization with multiplex SBE reactions in a single sample screened in one analysis. By screening for particular SNP genotypes, this assay can be used to de...