PCR Analysis of Chloroplast Double-Strand Break (DSB) Repair Products Induced by I-CreII in Chlamydomonas and Arabidopsis

Homing endonuclease I-CreII has been used to study the consequences and repair of a double-strand break (DSB) in the chloroplast genome of Chlamydomonas and Arabidopsis . Since I-CreII is from a mobile psbA intron of Chlamydomonas , it cleaves the psbA gene of an intronless-psbA strain of Chlamydomonas . And it cleaves specifically in the psbA gene of Arabidopsis , which is naturally intronless. We have shown further that most of the repair products of an I-CreII-induced break in chloroplast DNA can be defined by PCR analysis with total nucleic acids and the appropriate primers. Here, we provide protocols for small-scale preparation of nucleic acids from Chlamydomonas and Arabidopsis , as well as guidelines for the subsequent PCR analysis.