METHOD AND REAGENT COMBINATION FOR DETERMINING NUCLEOTIDE SEQUENCES

摘要

The invention provides for detection of changes in a nucleotide sequence based on the determination of the nucleotide sequence over the region of interest. The first step of the process involves obtaining an amplified DNA sample in which at least one attachment moiety has been introduced into at least one strand of a target polynucleo- tide by a modified polymerase chain reaction. This can be accomplished by synthesizing DNA using the target nucleic acid polymer as a template and using at least one amplifica- tion primer, which includes an attachment moiety bonded to the oligonucleotide. The resulting amplification mixture is repeatedly cycled between a polymerization condition and a denaturation condition occurs for a number of cycles suffi- cient to produce copies of the target nucleic acid polymer in a quantity suitable for use in a DNA sequencing procedure to form an amplified sample comprising copies of the target nucleic acid sequence bonded to the attachment moiety. After amplification, the strands of target nucleic acid polymer carrying an attachment moiety are captured using a solid matrix coated with an attachment site to which the attachment moiety or a modification thereof can bind. In this way, the strands of target nucleic acid become bound to the solid matrix and can be readily separated with the solid matrix from unbound reactants for sequencing.