Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

Lu, Ching-You; Tso, Dan-Ju; Yang, Tom P.; Jong, Yuh‐Jyh; Wei, Yau‐Huei

doi:10.1016/s0009-8981(01)00809-9

Cited by 30 publications

(23 citation statements)

References 13 publications

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“…Originally, RFLP had the disadvantage of the possibility of ambiguous bands on gels, but by using the bioanalyzer, accu- (11) found that variations in fragment sizes obtained among bioanalyzer chips were less than 10 bp, and this is in agreement with our finding of a variation of 5 bp or less. By using the data produced following digestion with several enzymes separately, we increased the discriminatory power of the technique.…”

Section: Discussionsupporting

confidence: 90%

“…Nachamkin et al (16) analyzed the flagellin gene of Campylobacter jejuni but found problems resolving fragments differing by 8 to 20 bp using the bioanalyzer compared to an agarose gel. In contrast, Lu et al (11) studied mutations in human mitochondrial DNA by RFLP and found that the bioanalyzer showed better reproducibility than the conventional method. In this study, we demonstrate that the Agilent 2100 bioanalyzer is a useful tool for obtaining accurate and reproducible-enough sizing of RFLP fragments for molecular typing of S. pneumoniae.…”

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confidence: 98%

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Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

et al. 2007

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Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.Streptococcus pneumoniae is an important human pathogen with a clonal population structure (4). Molecular typing has become increasingly important in order to understand the evolution of this pathogen and to trace clones with special traits such as antibiotic resistance. Several methods for genotypic analysis of S. pneumoniae are currently in use. The "gold standards" are pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). In PFGE, the whole genome is digested with a restriction endonuclease and run slowly through an agarose gel with an alternating current (10). The fragment banding pattern produced is used to characterize the strain. PFGE has the advantage of high discrimination between strains but is relatively laborious, time-consuming, and costly and tends to be used to compare strains within a laboratory rather than between laboratories because it involves the comparison of banding patterns rather than absolute values. MLST allows a direct comparison of genotypes by comparing sequences within several housekeeping genes (4). This technique has proven extremely useful in making comparisons between strains not only within a laboratory but also internationally by using a Web-based database. Sequencing, however, is currently expensive and time-consuming.Restriction fragment length polymorphism (RFLP) analysis is often used on a PCR amplification product that is subjected to endonuclease digestion followed by comparison of the resulting banding patterns produced on an agarose gel. This technique has been performed on coding regions such as the capsule genes (1) and penicillin-binding protein genes (12). It has some of the sho...

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Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

et al. 2007

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“…The chip is designed for the analysis of DNA fragments ranging from 100 to 7,500 bp in length. The procedure was illustrated in a recent publication (Lu et al 2002). In short, the DNA fragments within each sample are separated and labelled within a proprietary gel matrix containing a fluorogenic dye.…”

Section: Methodsmentioning

confidence: 99%

Microchip capillary electrophoresis-based genetic comparison of closely related cyathostomin nematode parasites of horses using randomly amplified polymorphic DNA polymerase chain reaction

Posedi

Drögemüller²,

Schnieder³

et al. 2004

Parasitol Res

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The microchip-based capillary electrophoresis technology represents a valuable recent development for the analysis of complex DNA banding patterns. We have used this technology for the differentiation of the closely related cyathostomin species Cylicocyclus elongatus and C. insigne from the horse. We found that the Agilent 2100 bioanalyser in combination with the DNA 7500 Lab Chip were suited to perform a phylogenetic DNA fingerprinting analysis of the parasite species studied. The analysis of the electrophoretic data was optimised and it was possible to resolve a phylogenetic tree where all 12 individual worms of the two Cylicocyclus species studied were assigned to their species as determined by microscopic identification based on morphological traits. Thus, our data indicated that the procedure described here provides an additional powerful tool that can be employed for species delineation of closely related strains or species, such as the two taxa of Cylicocyclus investigated in the present study. Furthermore, by determining the second internal transcribed spacer region of three and nine individual worms for C. elongatus and C. insigne, respectively, low intraspecific variations of only up to 0.3% were demonstrated.

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“…Publication Number 5988-7650EN. 2002) (5), a pool of the RNA samples described above or a single DNA sample (RNA-Seq library pool) was loaded as technical replicates in each well of duplicate chips for all three chip types tested. Library length and RIN were consistent between all chips (Figure 2).…”

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Bioanalyzer Chips can be Used Interchangeably For Many Analyses Of DNA or RNA

2016

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The Agilent 2100 Bioanalyzer enables small-scale gel electrophoretic separation of nucleic acids on a microfluidic chip; however, a shortage of chips and an excess of reagents are common issues. Here we explore the compatibility of two commonly used Bioanalyzer reagents with three Bioanalyzer chip types. Microfluidic electrophoretic separation of DNA and RNA using DNA High Sensitivity and RNA 6000 Nano reagents, respectively, was successfully performed on multiple chip types following the assay-specific protocols. For RNA quality and next-generation sequencing (NGS) library length estimation, the Bioanalyzer chips we tested can be used interchangeably. These findings will be valuable for any laboratory using the Agilent Bioanalyzer in a shared facility.

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Detection of DNA mutations associated with mitochondrial diseases by Agilent 2100 bioanalyzer

Cited by 30 publications

References 13 publications

Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

Use of the Agilent 2100 Bioanalyzer for Rapid and Reproducible Molecular Typing of Streptococcus pneumoniae

Microchip capillary electrophoresis-based genetic comparison of closely related cyathostomin nematode parasites of horses using randomly amplified polymorphic DNA polymerase chain reaction

Bioanalyzer Chips can be Used Interchangeably For Many Analyses Of DNA or RNA

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