Electrophoresis of DNA in human genetic diagnostics – state‐of‐the‐art, alternatives and future prospects

Gödde, René; Akkad, Denis-Amer; Arning, Larissa; Dekomien, Gabriele; Herchenbach, Julia; Kunstmann, Erdmute; Meins, Moritz; Wieczorek, Stefan; Epplen, Jörg T.; Hoffjan, Sabine

doi:10.1002/elps.200500675

Cited by 26 publications

(13 citation statements)

References 81 publications

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“…1 Currently, capillary electrophoresis has become widely used in molecular diagnostic laboratories principally because of its single base resolution and ability to automate both the loading and analysis. 2,3 Routine retrieval of molecules after electrophoretic separation is less straightforward. Several methods have been developed to retrieve and purify DNA fragments from ethidium bromide-stained agarose gels for subsequent analyses, such as cloning or DNA sequencing.…”

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confidence: 99%

A Molecular Fraction Collecting Tool for the ABI 310 Automated Sequencer

Lin

Rich

Shipley

et al. 2007

The Journal of Molecular Diagnostics

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Several methods exist to retrieve and purify DNA fragments after agarose or polyacrylamide gel electrophoresis for subsequent analyses. However, molecules present in low concentration and molecules similar in size to their neighbors are difficult to purify. Capillary electrophoresis has become popular in molecular diagnostic laboratories because of its automation, excellent resolution, and high sensitivity. In the current study, the ABI Prism 310 Genetic Analyzer was reconfigured into a fraction collector by adapting the standard gel block to accommodate a collection tube at the distal end of capillary. The time to collect the desired peaks was estimated by extrapolating from standard capillary electrophoresis using the original gel block. Fraction collection from a mixture of DNA fragments amplified from wild type and several internal tandem duplication mutations of the FMS-like tyrosine kinase 3 (Flt3) gene yielded highly purified DNA fragments containing internal tandem duplication mutations and predictable electrokinetics using the reconstructed gel block. The reconfigured instrument could successfully isolate DNA amplicons from extremely low-amplitude peaks (110 relative fluorescent units), which were undetectable using polyacrylamide gel electrophoresis. In addition, we successfully isolated bands that were only three bases apart that comigrated on polyacrylamide gel electrophoresis. DNA sequencing was used to confirm that the correct peaks were recovered at sufficient purity. (J Mol Diagn 2007, 9

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confidence: 99%

A Molecular Fraction Collecting Tool for the ABI 310 Automated Sequencer

Lin

Rich

Shipley

et al. 2007

The Journal of Molecular Diagnostics

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“…CE is a well-established, sensitive, and specific tool for the electrophoretic separation of a wide variety of biomolecules including DNA molecules [24][25][26][27]. Recently, the HDA system was developed as a 12-channel CE instrument to provide a simple, rapid, and reliable alternative to genetic quantification [28,29].…”

Section: Discussionmentioning

confidence: 99%

Use of multiplex PCR and CE for gene dosage quantification and its biomedical applications for SMN, PMP22, and α‐globin genes

et al. 2007

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Many genetic diseases are caused by the presence of point mutations, small insertions, and deletions in respective genes, and the number of diseases known to be caused by deletions and duplications involving large DNA genomes is increasing. These changes lead to underexpression or overexpression of the gene, according to changes in gene dosage. The methods for the detection of point mutations, small insertions, and deletions are well established, but the detection of larger genomic deletions or duplications is more difficult. Due to the lack of efficient and technically feasible protocols for gene dosage quantification, we describe a diagnostic protocol employing a combination of available methods. The efficient and accurate gene dosage quantification platform is combined with multiplex PCR and CE, and applied to detect dosages of several genes, including SMN, PMP22, and alpha-globin genes. The reliability of this novel methodology shows that it is a relatively speedy and low-cost procedure and a significant tool for genetic diagnosis. Its sensitivity and specificity for identifying deletion and duplication genotypes approach 100%. Moreover, once we establish this powerful system, we will further apply this technique to the rapid detection of trisomy syndromes and microdeletion syndromes, including trisomy 13, Down syndrome, DiGeorge syndrome, and others.

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“…It has been used for the analysis of a large variety of compound classes, such as peptides and proteins [1,2], DNA and RNA [3][4][5], lipids [6], low-molecular-weight drugs [7,8], and even inorganic ions [9,10]. Advantages of CE include high efficiency and speed, and low sample and solvent consumption.…”

Section: Introductionmentioning

confidence: 99%

Design and applications of coupled SPE‐CE

et al. 2007

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CE suffers from an inherent low concentration sensitivity. Analyte detection limits can be improved by combining CE with SPE. This paper presents an overview of coupled SPE-CE systems that have been reported in literature. Attention is paid to fundamental aspects of coupling SPE and CE, as well as to important SPE requirements. Interfaces for inline and online coupling with CE are critically discussed, and their mode of operation is outlined. Advantages and limitations of the interfaces are discussed and typical examples are selected. Finally, some future developments are discussed.

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Electrophoresis of DNA in human genetic diagnostics – state‐of‐the‐art, alternatives and future prospects

Cited by 26 publications

References 81 publications

A Molecular Fraction Collecting Tool for the ABI 310 Automated Sequencer

A Molecular Fraction Collecting Tool for the ABI 310 Automated Sequencer

Use of multiplex PCR and CE for gene dosage quantification and its biomedical applications for SMN, PMP22, and α‐globin genes

Design and applications of coupled SPE‐CE

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