CELL-FREE ASSAY AND IN VIVO METHOD FOR PLANT GENETIC REPAIR USING CHLOROPLAST LYSATE

摘要

An in vivo or in vitro cell-free method for genetic repair of mutation in plastid genes has been found which consists of (1) reacting a plasmid which contains a specific mutation (point mutation or frameshift mutation) of interest, a chimeric RNA/DNA oligonucleotide or a modified single stranded oligonucleotide which is believed to contain the genetic code for correcting the plastid gene mutation, and a chloroplast extract taken from the plant of interest, and (2) determining the success of gene conversion using a genetic readout system. A cell-free assay is disclosed by which the enzymatic capacity of chloroplast extracts to direct gene repair such as corrections to both point mutations and frameshift mutations can be determined. This assay method also enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA repair pathways.