PCR analysis of chloroplast double-strand break (DSB) repair products induced by I-CreII in Chlamydomonas and Arabidopsis

Methods Mol Biol. 2014:1123:77-86. doi: 10.1007/978-1-62703-968-0_6.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics*
  • Arabidopsis / metabolism
  • Chlamydomonas / genetics*
  • Chlamydomonas / metabolism
  • Chloroplasts / genetics*
  • Chloroplasts / metabolism
  • DNA Breaks, Double-Stranded*
  • DNA Repair*
  • DNA, Chloroplast / genetics*
  • DNA, Chloroplast / metabolism
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Polymerase Chain Reaction* / methods

Substances

  • DNA, Chloroplast
  • Deoxyribonucleases, Type II Site-Specific