The Experts below are selected from a list of 15600 Experts worldwide ranked by ideXlab platform
James P. Landers - One of the best experts on this subject based on the ideXlab platform.
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An integrated sample-in-answer-out microfluidic chip for rapid human identification by STR Analysis
Lab on a Chip, 2014Co-Authors: Delphine Le Roux, James P. Landers, Brian E. Root, Jeffrey A. Hickey, Orion N. Scott, Anchi Tsuei, Jingyi Li, David J. Saul, Luc Chassagne, Philippe De MazancourtAbstract:A fully integrated microfluidic chip for human identification by short tandem repeat (STR) Analysis that includes a unique enzymatic liquid preparation of the DNA, microliter non-contact PCR, and a polymer that allows a high-resolution separation within a compact microchip footprint has been developed. A heat-activated enzyme that digests biological materials is employed to generate the target yield of DNA from a buccal swab or FTA paper. The microfluidic architecture meters an aliquot of the liberated DNA and mixes it with the PCR reagents prior to non-contact IR-mediated PCR amplification. The products of PCR amplification are mixed with a sizing standard (ladder) and the 18-plex STR amplicons are separated in an effective length (L eff) of just 7 cm. The development, optimization and integration of each of these processes within the microfluidic chip are described. The device is able to generate genetic profiles in approximately 2 hours that match the profiles from the conventional processes performed using separate conventional inSTRuments. Analysis is performed on a single plastic microchip with a size similar to that of a 96-well plate and only a few mm thick with no pretreatment of any of the functional domains. This is significant advancement in terms of ease of fabrication over glass microdevices or polymeric systems assembled from multiple components. Consequently, this fully integrated sample-in-answer-out microchip is an important step toward generation of a rapid micro-total Analysis system for point-of-collection human identification based on genetic Analysis.
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A valveless microfluidic device for integrated solid phase extraction and polymerase chain reaction for short tandem repeat (STR) Analysis
The Analyst, 2011Co-Authors: Kristin A. Hagan, Carmen R. Reedy, Joan M. Bienvenue, Alison H. Dewald, James P. LandersAbstract:A valveless microdevice has been developed for the integration of solid phase extraction (SPE) and polymerase chain reaction (PCR) on a single chip for the short tandem repeat (STR) Analysis of DNA from a biological sample. The device consists of two domains—a SPE domain filled with silica beads as a solid phase and a PCR domain with an ∼500 nL reaction chamber. DNA from buccal swabs was purified and amplified using the integrated device and a full STR profile (16 loci) resulted. The 16 loci Identifiler® multiplex amplification was performed using a non-contact infrared (IR)-mediated PCR system built in-house, after syringe-driven SPE, providing an ∼80-fold and 2.2-fold reduction in sample and reagent volumes consumed, respectively, as well as an ∼5-fold reduction in the overall Analysis time in comparison to conventional Analysis. Results indicate that the SPE-PCR system can be used for many applications requiring genetic Analysis, and the future addition of microchip electrophoresis (ME) to the system would allow for the complete processing of biological samples for forensic STR Analysis on a single microdevice.
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AOTF-based multicolor fluorescence detection for short tandem repeat (STR) Analysis in an electrophoretic microdevice
Lab on a chip, 2008Co-Authors: James M. Karlinsey, James P. LandersAbstract:An acousto-optic tunable filter (AOTF) has been used to perform multicolor fluorescence detection for four and five-color short tandem repeat (STR) Analysis on glass microchips. Matrix files were initially generated by collecting and comparing the laser-induced fluorescence emission of the labels specific to a particular STR kit, and raw data was processed to remove spectral overlap. The AmpFlSTR kits used in this work include Profiler Plus and COfiler, which are four-color kits used in tandem to address the core STR loci, as well as the five-color Identifiler kit, which contains each of the loci. In contrast to previous reports on multicolor detection for STR Analysis on microchips, this detection system is characterized by a single filter and detector, and reports the first five-color genotyping application on-chip. This capability matches the portability and reduced scale of the microchip with the state-of-the-art in multicolor STR Analysis kits.
Bart J. Blankers - One of the best experts on this subject based on the ideXlab platform.
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Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method
Investigative genetics, 2013Co-Authors: Bas Kokshoorn, Bart J. BlankersAbstract:In a recent contribution to this journal Grisedale and Van Daal concluded that a single STR Analysis of all available template DNA is to be preferred over replicate analyses and a consensus approach when analyzing low template DNA samples. A single STR Analysis approach does not allow for an assessment of the validity of the resulting DNA profile. We argue that the use of replicate amplifications is the best way to objectively quantify the extent of the stochastic variation in the data. By applying consensus methodology and/or a probabilistic model, the interpretation of the data will therefore be more objective and reliable.
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Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method
Investigative Genetics, 2013Co-Authors: Bas Kokshoorn, Bart J. BlankersAbstract:In a recent contribution to this journal Grisedale and Van Daal concluded that a single STR Analysis of all available template DNA is to be preferred over replicate analyses and a consensus approach when analyzing low template DNA samples. A single STR Analysis approach does not allow for an assessment of the validity of the resulting DNA profile. We argue that the use of replicate amplifications is the best way to objectively quantify the extent of the stochastic variation in the data. By applying consensus methodology and/or a probabilistic model, the interpretation of the data will therefore be more objective and reliable. Please see related article: http://www.investigativegenetics.com/content/3/1/14
Johan Fevery - One of the best experts on this subject based on the ideXlab platform.
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Identification of HepG2 variant cell lines by short tandem repeat (STR) Analysis
Molecular and Cellular Biochemistry, 2003Co-Authors: Jos F. Van Pelt, Ronny Decorte, Johan FeveryAbstract:In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.
Bas Kokshoorn - One of the best experts on this subject based on the ideXlab platform.
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Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method
Investigative genetics, 2013Co-Authors: Bas Kokshoorn, Bart J. BlankersAbstract:In a recent contribution to this journal Grisedale and Van Daal concluded that a single STR Analysis of all available template DNA is to be preferred over replicate analyses and a consensus approach when analyzing low template DNA samples. A single STR Analysis approach does not allow for an assessment of the validity of the resulting DNA profile. We argue that the use of replicate amplifications is the best way to objectively quantify the extent of the stochastic variation in the data. By applying consensus methodology and/or a probabilistic model, the interpretation of the data will therefore be more objective and reliable.
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Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method
Investigative Genetics, 2013Co-Authors: Bas Kokshoorn, Bart J. BlankersAbstract:In a recent contribution to this journal Grisedale and Van Daal concluded that a single STR Analysis of all available template DNA is to be preferred over replicate analyses and a consensus approach when analyzing low template DNA samples. A single STR Analysis approach does not allow for an assessment of the validity of the resulting DNA profile. We argue that the use of replicate amplifications is the best way to objectively quantify the extent of the stochastic variation in the data. By applying consensus methodology and/or a probabilistic model, the interpretation of the data will therefore be more objective and reliable. Please see related article: http://www.investigativegenetics.com/content/3/1/14
Jingliang Cheng - One of the best experts on this subject based on the ideXlab platform.
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a case of usher syndrome type iia caused by a rare ush2a homozygous frameshift variant with maternal uniparental disomy upd in a chinese family
Journal of Cellular and Molecular Medicine, 2020Co-Authors: Jiewen Fu, Shiyi Shen, Jingliang Cheng, Hongbin Lv, Junjiang FuAbstract:Usher syndrome encompasses a group of genetically and clinically heterogeneous autosomal recessive disorders with hearing deficiencies and retinitis pigmentosa. The mechanisms underlying the Usher syndrome are highly variable. In the present study, a Chinese family with Usher syndrome was recruited. Whole exome sequencing (WES), Sanger sequencing, homozygosity mapping, short tandem repeat (STR) Analysis and segregation Analysis were performed. Functional domains of the pathogenic variant for USH2A were analysed. We identified a homozygous frameshift variant c.99_100insT (p.Arg34Serfs*41) in the USH2A gene in the proband that showed discordant segregation in the father. Further homozygosity mapping and STR Analysis identified an unusual homozygous variant of proband that originated from maternal uniparental disomy (UPD). The p.Arg34Serfs*41 variant produced a predicted truncated protein that removes all functional domains of USH2A. The variant was not included in the 1000 Human Genomes Project database, ExAC database, HGMD or gnomAD database, but was included in the ClinVar databases as pathogenic. Although USH2A is an autosomal recessive disease, the effects of UPD should be informed in genetic counselling since the recurrence risk of an affected child is greatly reduced when the disease is due to the UPD mechanism. To test potential patients, WES, combined with STR Analysis and homozygosity mapping, provides an accurate and useful STRategy for genetic diagnosis. In summary, our discoveries can help further the understanding of the molecular pathogenesis of Usher syndrome type IIA to advance the prevention, diagnosis and therapy for this disorder.
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Evaluation genotypes of cancer cell lines HCC1954 and SiHa by short tandem repeat (STR) Analysis and DNA sequencing.
Molecular biology reports, 2018Co-Authors: Jingliang Cheng, Xiaoyan Liu, Chunli Wei, Xiaoli ZhengAbstract:Cancer cell lines are used worldwide in biomedical researches, and data interpretation solely depends on unambiguous attribution of those respective cell lines to its original sources. Approximately one-third of all cell lines have an origin other than that assumed, leading to invalid results. It is necessary to characterize the origin of cell lines. Short-tandem-repeat (STR) fingerprinting (DNA fingerprinting) is the method for characterization of genetic identity in cultured cell lines under certain experimental conditions. We showed the fingerprinting profiles in a summed and unidentified human cancer cell line comparison to HCC1954 cell line, revealing marked alterations in DNA fingerprinting profiles up to fourteen STR loci from 16 loci. Furthermore, Sanger DNA sequencing showed no c.3140A > G heterozygous mutation in the PIK3CA gene of this suspected HCC1954 cell line. In addition, we showed the fingerprinting profiles in an unidentified cancer cell line comparison to SiHa cervical cell line, revealing same DNA fingerprinting profiles. In conclusion, we have successfully authenticated and identified both suspected HCC1954 and SiHa cell lines by STR Analysis and DNA sequencing. STR Analysis combined DNA sequencing may be very useful to evaluate genotypes of cancer cell lines in our cancer studies, as well as in judicial authentication and forensic sciences.
