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Multiplex high-throughput solid-phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection
KTH, Superseded Departments, Chemistry.
2002 (English)In: Electrophoresis, ISSN 0173-0835, E-ISSN 1522-2683, Vol. 23, no 10, 1467-1472 p.Article in journal (Refereed) Published
Abstract [en]

Minisequencing, solid-phase single-nucleotide primer extension reaction, is a robust method for performing multiplex single-nucleotide polymorphism (SNP) analysis. We have combined this technology with capillary gel electrophoresis in a multicapillary format, using liquid core waveguide (LCW) fluorescence detection. Polymerase chain reaction (PCR) amplification of multiple DNA targets is performed with one primer for each target biotinylated. Separation of the complementary strands, minisequencing and washing steps are carried out using streptavidin-coated magnetic beads. Dideoxynucleotides analogues labelled with different fluorophores are used for the extension of the minisequencing primers. The extended oligonucleotides, the length of which defines the position on the target and the color the identity of the polymorphism, are then separated in a gel-filled array of capillaries, coated on the outside with a layer of a fluoropolymer to provide the liquid core waveguide characteristics. The technology has a potential for extremely high throughputs when a combination of multiplex PCR-minisequencing is used together with a large array of capillaries, four-color detection and high-speed separation.

Place, publisher, year, edition, pages
2002. Vol. 23, no 10, 1467-1472 p.
Keyword [en]
capillary array, liquid core waveguide, minisequencing, single-nucleotide polymorphism, tof mass-spectrometry, single-nucleotide polymorphisms, dna-sequence, gene, identification, extension, products
URN: urn:nbn:se:kth:diva-21595ISI: 000175992800013OAI: diva2:340293
QC 20100525Available from: 2010-08-10 Created: 2010-08-10Bibliographically approved

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