Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

• Published in: Nucleic acids research 2002-03, Vol.30 (5), p.e19-e19
• Main Authors: Tong, Anthony K, Ju, Jingyue
• Format: Article
• Language: English
• Subjects: Biotinylation; combinatorial fluorescence energy transfer; Dideoxynucleosides - chemistry; dideoxynucleotides; DNA Primers - chemistry; DNA-Directed DNA Polymerase - chemistry; Electrophoresis - methods; Energy Transfer; Fluorescence; Fluorescent Dyes - chemistry; Humans; NAR Methods Online; Polymerase Chain Reaction; Polymorphism, Single Nucleotide; Retinoblastoma Protein - genetics; Sequence Analysis, DNA - methods
• ISSN: 1362-4962; 0305-1048; 1362-4962
• DOI: 10.1093/nar/30.5.e19

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3'-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3'-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.