INTENSITY ENCODED DNA SEQUENCING

DNA SEQUENCING TYPICALLY REQUIRES FOUR PARALLEL REACTIONS RUN ON FOUR PARALLEL LANES OF AN ELECTROPHORESIS GEL. TOGETHER WITH GEL INTERPRETATION, THIS REPRESENTS MOST OF THE LABOR ONCE THE TEMPLATE DNA IS PREPARED. WE WILL DEVELOP A SIMPLIFIED DNA SEQUENCING METHOD USING A SINGLE REACTION AND A SINGLE LANE OF A GEL. THE SEQUENCE INFORMATION WILL BE ENCODED IN THE INTENSITIES OF THE BANDS. THIS INTENSITY-ENCODED SEQUENCING (IES) METHOD TAKES ADVANTAGE OF THREE RECENT ADVANCES CURRENTLY BEING MARKETED AND DEVELOPED AT UNITED STATES BIOCHEMICAL CORPORATION (USB). ONE IS SEQUENCE VERSION 2.0, A PATENTED ENZYME WHICH GENERATES EXCEPTIONAL SEQUENCING RESULTS. THE SECOND IS THE USE OF MN2+ AND INORGANIC PYROPHOSPHATASE WITH SEQUENASE. THESE MAKE CHAIN TERMINATION SEQUENCE-INDEPENDENT, GIVING UNIFORM BAND INTENSITIES. THUS, ALL FOUR DIDEOXY-NUCLEOTIDES CAN BE INCLUDED AT DIFFERENT CONCENTRATIONS IN A SINGLE REACTION; BAND INTENSITY DEPENDS ON THE CONCENTRATION, SO THE NUCLEOTIDE CAN BE DETERMINED FROM THE BAND INTENSITY. FINALLY, THE GELS ARE READ USING AN INEXPENSIVE PRECISION OPTICAL SCANNER TO BE INTRODUCED BY USB IN EARLY 1991. IES IS SIMPLE AND INEXPENSIVE ENOUGH TO BE RUN IN ANY LABORATORY THAT DOES SEQUENCING. COMMERCIAL PRODUCTS WILL INCLUDE A REAGENT KIT FOR RUNNING IES REACTIONS AND SOFTWARE TO INTERPRET GELS.