Accurate Direct PCR with Arabidopsis and rice

Abstract

AbstractMethods for PCR that avoid costly and time consuming plant DNA purification have not been widely adopted, partly because their efficacy is unclear. Here, we compare different sampling methods for Direct PCR in Arabidopsis and rice. CutTip, stabbing a pipette tip into a plant organ and depositing the tip of the pipette tip into the reaction buffer, yielded high accuracy for genotyping and detection of CRISPR-induced mutations. This did not require visible tissue fragments in the reactions. We demonstrate the usefulness of this method in sampling many locations within a single plant to identify a rare CRISPR-mutated sector. These methods are simple, inexpensive and can help address the challenge of genotyping and genome editing at different scales with high accuracy. The methods also simplify the application of PCR in the field.