DNA sequencing with solid-phase-capturable dideoxynucleotides and energy transfer primers.
False terminations occurring in fluorescent dye-primer DNA sequencing, and nonsequencing primer extension DNA fragments generated in dye-terminator sequencing cause background noise in fluorescent electropherograms, leading to errors in sequence determination. We describe here a DNA sequencing chemistry that produces accurate and clean sequencing data on a fluorescent DNA sequencer, eliminating the false terminations and background noise. The procedure involves coupling fluorescence energy transfer (ET) primers that produce high fluorescent signals with solid-phase-capturable biotinylated dideoxynucleotides to generate Sanger DNA sequencing fragments. After the sequencing reaction,the DNA extension fragments that carry a biotin at the 3' end are captured with streptavidin-coated magnetic beads, while the other components in the sequencing reaction are washed away. Only pure DNA extension products terminated by the biotinylated dideoxynucleotides are released from the magnetic beads and are loaded onto a sequencing gel to produce accurate sequencing data.
Ju
Jingyue
J
Laboratory of DNA Sequencing and Chemical Biology, Columbia Genome Center, Russ Berrie Medical Science Pavilion, Columbia University College of Physicians and Surgeons, 1150 St. Nicholas Avenue, New York, NY 10032, USA. dj222@columbia.edu
D001483:Base Sequence; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D015224:Dideoxynucleosides / Q000737:chemistry* / Q000378:metabolism; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer; D008863:Microspheres; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin / Q000378:metabolism; D013698:Templates, Genetic
D017931:DNA Primers; D015224:Dideoxynucleosides; D001710:Biotin; D004247:DNA; D019809:Streptavidin