title: Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides.
abstract: 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.
authors.affiliation: Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Columbia University College of Physicians and Surgeons, 1150 St Nicholas Avenue, New York, NY 10032, USA.
authors.forename: Anthony K
authors.initials: AK
authors.lastname: Tong
authors.forename: Jingyue
authors.initials: J
authors.lastname: Ju
mesh_terms: D019921:Biotinylation; D017931:DNA Primers / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D015224:Dideoxynucleosides / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods; D004735:Energy Transfer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D016160:Retinoblastoma Protein / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D017931:DNA Primers; D015224:Dideoxynucleosides; D005456:Fluorescent Dyes; D016160:Retinoblastoma Protein; D004259:DNA-Directed DNA Polymerase