IMINOBIOTINYLATED DIDEOXYNUCLEOTODES FPR DNA SEQUENCING

A VARIETY OF DIFFERENT APPROACHES ARE BEING TAKEN TO INCREASE THE SPED AND DECREASING THE SPEED AND DECREASE THE COST OF DNA SEQUENCING. ONE OF THE MAJOR CURRENT SEQUENCING TECHNOLOGIES IS THE DIDEOXYNUCLEOTIDE CHAIN-TERMINATING APPROACH ORIGINALLY DEVELOPED BY SANGER. WE WILL USE A VARIANT OF THIS APPROACH IN WHICH THE DIDEOXYNUCLEOTIDES WILL BE MODIFIED WITH AN IMINOBIOTIN GROUP. IMINOBIOTIN BINDS STREPTAVIDIN ABOVE PH5.0, BUT LOSES ITS AFFINITY BELOW PH5.0. AFTER THE SEQUENCING REACTION, THE LABELED DNA LADDER CAN BE SEPARATED, USING STREPTAVIDIN, FROM THE TEMPLATE DNA AND OTHER RACTION COMPONENTS COMPONENTS PRIOR TO GEL ELECTROPHORESIS. THIS WILL RESULT IN VERY CLEAN SAMPLES WITH SMALL VOLUMES WHICH WILL YIELD SHARPER BANDS, IMPROVED SIGNAL TO NOISE RATIOS AND CONSEQUENTLY THE ABILITY TO READ MORE DNA SEQUENCE DATA PER GEL. THIS WILL FACILITATE DIRECT SEQUENCING OF LARGE TEMPLATES SUCH AS COSMIDS OR YACS WHERE A LARGE TEMPLATE MASS IS NEEDED TO HAVE A SUFFICIENT NUMBER OF TEMPLATE MOLECULES. THIS WILL ALSO FACILITATE SEQUENCING USING CAPILLARY ELECTROPHORESIS WHERE SMALL SAMPLE VOLUMES ARE CRITICAL. AN ADDITIONAL ADVANTAGE IS THAT ONLY LEGITMATELY TERMINATED LABELED PRODUCTS WILL BE SELECTED. LABELED PRODUCTS WHICH TERMINATE DUE TO REGIONS OF TEMPLATE SECONDARY STRUCTURE OR OTHER ILLGITIMATE EVENTS WILL NOT CONTAIN AN IMINOBIOTIN AND THUS, WILL NOT BE SELECTED. THIS WILL REDUCE CROSS LANE BANDING ARTIFACTS AND BACKGROUND NOISE. THIS APPROACH WILL ALSO IMPROVE THE RELIABILITY OF SEQUENCING RNA WITH REVERSE TRANSCRIPASE SINCE THIS ENZYME EXHIBITS HIGH LEVELS OF NONSPECIFIC TERMINATIONS.