Premium
Multiplex high‐throughput solid‐phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection
Author(s) -
Curcio Mario,
Stålhandske Per,
Lindberg Peter,
Roeraade Johan
Publication year - 2002
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(200205)23:10<1467::aid-elps1467>3.0.co;2-e
Subject(s) - primer extension , multiplex , biotinylation , capillary electrophoresis , primer (cosmetics) , streptavidin , oligonucleotide , polymerase chain reaction , multiplex polymerase chain reaction , microbiology and biotechnology , dna , chromatography , biology , chemistry , genetics , gene , biotin , base sequence , organic chemistry
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.