The Experts below are selected from a list of 4074 Experts worldwide ranked by ideXlab platform
Gustav Gaudernack - One of the best experts on this subject based on the ideXlab platform.
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Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis.
Mutation research, 2003Co-Authors: Jens Bjørheim, Torveig Weum Abrahamsen, Annette Torgunrud Kristensen, Gustav Gaudernack, Per O EkstrømAbstract:Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.
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automated constant denaturant capillary electrophoresis applied for detection of kras exon 1 mutations
BioTechniques, 2001Co-Authors: Jens Bjørheim, Per Olaf Ekstrom, Ellen Fossberg, Anne Lise Borresendale, Gustav GaudernackAbstract:In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 Genetic Analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.
Jens Bjørheim - One of the best experts on this subject based on the ideXlab platform.
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Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis.
Mutation research, 2003Co-Authors: Jens Bjørheim, Torveig Weum Abrahamsen, Annette Torgunrud Kristensen, Gustav Gaudernack, Per O EkstrømAbstract:Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.
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automated constant denaturant capillary electrophoresis applied for detection of kras exon 1 mutations
BioTechniques, 2001Co-Authors: Jens Bjørheim, Per Olaf Ekstrom, Ellen Fossberg, Anne Lise Borresendale, Gustav GaudernackAbstract:In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 Genetic Analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.
Per O Ekstrøm - One of the best experts on this subject based on the ideXlab platform.
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Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis.
Mutation research, 2003Co-Authors: Jens Bjørheim, Torveig Weum Abrahamsen, Annette Torgunrud Kristensen, Gustav Gaudernack, Per O EkstrømAbstract:Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.
Bruce Budowle - One of the best experts on this subject based on the ideXlab platform.
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Utility of the Ion S5™ and MiSeq FGx™ sequencing platforms to characterize challenging human remains
Legal Medicine, 2019Co-Authors: Kyleen Elwick, Sheree Hughes-stamm, Jonathan L King, Joseph Chang, Bruce BudowleAbstract:Abstract Often in missing persons’ and mass disaster cases the samples remaining for analysis are hard tissues such as bones, teeth, nails, and hair. These remains may have been exposed to harsh environmental conditions, which pose challenges for downstream genotyping. Short tandem repeat analysis (STR) via capillary electrophoresis (CE) is still the gold standard for DNA typing; however, a newer technology known as massively parallel sequencing (MPS) could improve upon our current techniques by typing different and more markers in a single analysis, and consequently improving the power of discrimination. In this study, bone and tooth samples exposed to a variety of DNA insults (cremation, embalming, decomposition, thermal degradation, and fire) were assessed and sequenced using the Precision ID chemistry and a custom AmpliSeq™ STR and iiSNP panel on the Ion S5™ System, and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™ system, as well as the GlobalFiler™ PCR Amplification Kit on the 3500™ Genetic Analyzer. The results demonstrated that using traditional CE-based genotyping performed as expected, producing a partial or full DNA profile for all samples, and that both sequencing chemistries and platforms were able to recover sufficient STR and SNP information from a majority of the same challenging samples. Run metrics including profile completeness and mean read depth produced good results with each system, considering the degree of damage of some samples. Most sample insults (except decomposed) produced similar numbers of alleles for both MPS systems. Comparable markers produced full concordance between the two platforms.
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internal validation of the globalfiler express pcr amplification kit for the direct amplification of reference dna samples on a high throughput automated workflow
Forensic Science International-genetics, 2014Co-Authors: Shahida Flores, Jonathan L King, Bruce Budowle, Jie SunAbstract:The GlobalFiler™ Express PCR Amplification Kit uses 6-dye fluorescent chemistry to enable multiplexing of 21 autosomal STRs, 1 Y-STR, 1 Y-indel and the sex-determining marker amelogenin. The kit is specifically designed for processing reference DNA samples in a high throughput manner. Validation studies were conducted to assess the performance and define the limitations of this direct amplification kit for typing blood and buccal reference DNA samples on various punchable collection media. Studies included thermal cycling sensitivity, reproducibility, precision, sensitivity of detection, minimum detection threshold, system contamination, stochastic threshold and concordance. Results showed that optimal amplification and injection parameters for a 1.2mm punch from blood and buccal samples were 27 and 28 cycles, respectively, combined with a 12s injection on an ABI 3500xL Genetic Analyzer. Minimum detection thresholds were set at 100 and 120RFUs for 27 and 28 cycles, respectively, and it was suggested that data from positive amplification controls provided a better threshold representation. Stochastic thresholds were set at 250 and 400RFUs for 27 and 28 cycles, respectively, as stochastic effects increased with cycle number. The minimum amount of input DNA resulting in a full profile was 0.5ng, however, the optimum range determined was 2.5-10ng. Profile quality from the GlobalFiler™ Express Kit and the previously validated AmpFlSTR(®) Identifiler(®) Direct Kit was comparable. The validation data support that reliable DNA typing results from reference DNA samples can be obtained using the GlobalFiler™ Express PCR Amplification Kit.
Mingchang Zhang - One of the best experts on this subject based on the ideXlab platform.
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Mutations of Desmoglein-2 in Sudden unexplained death in the chinese han population
Journal of Forensic Science and Medicine, 2019Co-Authors: Junyi Lin, Ziqin Zhao, Yulei Yang, Yiwen Shen, Mingchang ZhangAbstract:Sudden unexplained death (SUD) remains a puzzle in forensic medicine. Desmoglein-2 (DSG2) has been linked to arrhythmogenic right ventricular cardiomyopathy which may cause life-threatening ventricular arrhythmias and sudden death. Fatal arrhythmias resulting in sudden death also occur in the absence of morphologic cardiac abnormalities at autopsy. We hypothesized that DSG2 mutations may be responsible for certain Chinese SUD cases. We sequenced all 15 exons of DSG2 in DNA extracted from postmortem heart tissues of 25 Chinese patients dying from SUD. The primers were designed using the Primer Express 3.0 software. Direct sequencing for both sense and antisense strands was performed with a BigDye Terminator DNA sequencing kit on a 3130 Xl Genetic Analyzer. Mutation damage prediction was made using Mutation Taster, PolyPhen, and SIFT software. In 2 of 25 cases of Chinese SUD samples, two DSG2 heterozygous mutations (p.P927 L and p.T1070M) were identified, and one is probably damaging. We concluded that DSG2 mutations may be related to the occurrence of part of SUD cases in the Chinese Han population.
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Mutations of desmoglein-2 in sudden death from arrhythmogenic right ventricular cardiomyopathy and sudden unexplained death.
Forensic Science International, 2015Co-Authors: Mingchang Zhang, Ziqin Zhao, Joao Bosco Oliveira, Yiwen Shen, Aimin Xue, Allen P. BurkeAbstract:Desmoglein-2 (DSG2), a member of the desmosomal cadherin superfamily, has been linked to arrhythmogenic right ventricular cardiomyopathy (ARVC)which may cause life-threatening ventricular arrhythmias and sudden death. Fatal arrhythmias resulting in sudden death also occur in the absence of morphologic cardiac abnormalities at autopsy. We sequenced all 15 exons of DSG2 in DNA extracted from post-mortem heart tissues of 25 patients dying with ARVC and 25 from sudden unexplained death (SUD). The primers were designed using the Primer Express 3.0 software. Direct sequencing for both sense and antisense strands was performed with a BigDye Terminator DNA sequencing kit on a 3130 xl Genetic Analyzer. Mutation damage prediction was made using Mutation Taster, Polyphen and SIFT software. 2 DSG2 mutations (p. S1026Q fsX12, p. G678R)in two ARVC samples and 2 DSG2 mutations(p. E 896K, p. A858 V) in two SUD samples were identified, all the mutations were novel. We concluded that DSG2 mutations may not specific for ARVC and may be related to the fatal arrhythmic events even in patients with a morphological normal heart.
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PKP2 Mutations in Sudden Death From Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) and Sudden Unexpected Death With Negative Autopsy (SUDNA)
Circulation Journal, 2012Co-Authors: Mingchang Zhang, Fabio Tavora, Ziqin Zhao, Joao Bosco Oliveira, Marcello Franco, David Fowler, Ling Li, Allen BurkeAbstract:Background: Plakophilin2 (PKP2) is a desmosome-related protein with numerous armadillo repeats and has been linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). Fatal arrhythmias resulting in sudden death also occur in the absence of morphologic cardiac abnormalities at autopsy, and have been linked to ion channel mutations in a subset of cases, but so far not to PKP2. Methods and Results: We sequenced all 14 exons of PKP2 in DNA extracted from postmortem heart tissues of 25 patients dying from ARVC and 25 from sudden unexpected death with negative autopsy (SUDNA). The primers were designed using the Primer Express 3.0 software. Direct sequencing for both sense and antisense strands was performed with a BigDye Terminator DNA sequencing kit on a 3130XL Genetic Analyzer. Mutation damage prediction was made using Mutation Taster, Polyphen and SIFT software. In 6 of the 25 ARVC samples, 6 PKP2 mutations were identified, 4 of which were likely significant, and 3 of which were novel (p.N641del, p.L64PfsX22, p.G269R). In 6 of the 25 cases of SUDNA samples, 6 PKP2 mutations were identified, 3 of which were likely significant, and 4 of which were not previously described (p.P665S, p.Y217TfsX45, p.E540, p.S615T). Conclusions:PKP2 mutations are not specific for ARVC and may result in SUDNA. The link between ARVC and desmosomal mutations may not be causal but related to an association between defective desmosomal proteins and arrhythmias. (Circ J 2012; 76: 189-194)