Accurate and sensitive analysis of triplet repeat expansions by capillary electrophoresis

Olejniczak, Marta; Kozłowski, Piotr; Sobczak, Krzysztof; Krzyżosiak, Włodzimierz J.

doi:10.1002/elps.200410197

Cited by 10 publications

(8 citation statements)

References 25 publications

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“…While sieving gels offer high resolution of DNA fragments with different lengths, they are not designed for the separation of similar-length fragments based on differences in the sequence. Different approaches have therefore been developed for problems in biomedical or forensic analysis that involve resolution of DNA mutants or variants [8][9][10]. These approaches, including heteroduplex analysis, single stranded length conformational polymorphism, ACE, and probe-regulated simultaneous separation [11][12][13][14], have met with only limited success due to their reliance on sequence-dependent conformational differences.…”

Section: Introductionmentioning

confidence: 99%

Guanosine gel for sequence‐dependent separation of polymorphic ssDNA

et al. 2007

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The separation of fluorescent-labeled ssDNA fragments of equal length based on differences in sequence was achieved through the use of guanosine gels (G-gels) formed by guanosine-5'-monophosphate (GMP) in capillary gel electrokinetic chromatography (CGEKC) with LIF detection. Baseline resolution was achieved for homodimers and homopentamers of A, T, and C. G-gel CGEKC provided better resolution than CZE, MEKC, or a sieving gel in CGE. Resolution improved with increasing GMP, indicating that the interaction is linked to structural organization of the G-gel. Fluorescence intensity and anisotropy show that the order of interaction with G-gels is T>C>A. We then investigated four conformationally similar, polymorphic 76-mers with A/G substitutions that are utilized in forensic DNA typing. Resolution was achieved by CGEKC but not CZE or CGE. In CGEKC, the negatively charged G-gels and oligonucleotides electromigrate toward the positive inlet while being driven by EOF to the negative outlet. The net forward velocity is the greatest for oligonucleotides most closely associated with the slower, more cumbersome G-gel network. For the 76-mers, resolution increases with increasing difference in guanosine content between strands and, for a given difference, with increasing total guanosines in the strands.

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Section: Introductionmentioning

confidence: 99%

Guanosine gel for sequence‐dependent separation of polymorphic ssDNA

et al. 2007

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“…For the majority of AFLP studies, sizing of fragments is relative since the fluorochromes on the internal standard and the sample do not match. However, if exact sizing of fragments is required, as may be the case in some instances for clinical diagnostics, steps should be taken to include internal standards with matching fluorochromes [10].…”

Section: Repeatability Of Aflp Profile In the Preliminary Investigationmentioning

confidence: 99%

“…Dye labels on fragments can also influence migration rates. For instance, a fragment labeled with a fluorescein dye such as 5-carboxyfluorescein (FAM), 6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein (JOE) or 2,7',8-benzo-5'-fluoro-2',4,7-trichloro-5-carboxyfluorescein (NED) migrates faster through the POP-4 matrix, and hence appears smaller, than the same fragment labeled with one of the rhodamine dyes, 6-carboxyrhodamine (ROX) or 6-carboxy-N,N,N',N'-tetramethylrhodamine (TAMRA) [8,10].…”

Section: Introductionmentioning

confidence: 99%

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

Partis

Burns²,

Chiba

et al. 2007

Electrophoresis

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A simple amplified fragment length polymorphism (AFLP) model, using the bacteriophage lambda genome, was developed to test the reproducibility of this technique in an international comparative study. Using either non-selective or selective primers, nine fragments or subsets of two or three fragments, respectively, were predicted using in silico software. Under optimized conditions, all predicted fragments were experimentally generated. The reproducibility of the AFLP model was tested by submitting both "unknown" DNA template that had been restricted and ligated with AFLP linkers (R/L mixture) and corresponding primer pairs to nine laboratories participating in the study. Participants completed the final PCR step and then used either slab gel electrophoresis or CE to detect the AFLP fragments. The predicted fragments were identified by the majority of participants with size estimates consistently up to 3 base pair (bp) larger for slab gel electrophoresis than for CE. Shadow fragments, 3 bp larger than the predicted fragments, were often observed by study participants and organizers. The nine AFLP fragments exhibited relative intensities ranging from less than 3% to 22% and, apart from the two weakest fragments, with a % CV of 16 to 25. Fragments containing the highest guanine-cytosine (GC) content of 50-56% showed the greatest stability in the AFLP profiles.

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“…TRF_123nt to TRF_133nt. This window of 10 nucleotides was necessary because an absolute determination of the length of TRFs with capillary electrophoresis is currently not possible (Bruland et al 1999, Hahn et al 2001, Olejniczak et al 2005.…”

Section: Trf Pattern Analysesmentioning

confidence: 99%

Variations in pelagic bacterial communities in the North Atlantic Ocean coincide with water bodies

Hahnke¹,

Probian²,

Fuchs³

et al. 2013

Aquat. Microb. Ecol.

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Physical and chemical characteristics define oceanographic regions. The potential for a distinct biogeography of bacterial communities in these oceanic provinces was studied in epipelagic and upper mesopelagic water bodies of the North Atlantic Ocean by terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes and flow cytometry. Water samples from 67°N to 34°N along the 30°W meridian contained epipelagic populations of Synechococcus in the north and Prochlorococcus in the south. Bacterial communities were generally more diverse in phototrophic layers above the pycnocline. Communities significantly differed in the epipelagic zone along the latitudinal transect through the different oceanic provinces and between the epipelagic and the upper mesopelagic zone. Differences in the T-RFLP patterns coincided well with differences in the physico-chemical conditions of the sampling sites. Changes in bacterial communities were traced to characteristic terminal restriction fragments (TRFs). In silico assignments of phylogenetic groups to TRFs, e.g. populations of high-light and low-light ecotypes of Prochlorococcus, supported our T-RFLP analysis of bacterial communities. Distinct bacterial communities in water bodies of the North Atlantic Ocean hosted different bacterial populations, which may serve as biological markers for oceanic provinces.

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Accurate and sensitive analysis of triplet repeat expansions by capillary electrophoresis

Cited by 10 publications

References 25 publications

Guanosine gel for sequence‐dependent separation of polymorphic ssDNA

Guanosine gel for sequence‐dependent separation of polymorphic ssDNA

A study of comparability in amplified fragment length polymorphism profiling using a simple model system

Variations in pelagic bacterial communities in the North Atlantic Ocean coincide with water bodies

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