Genomic DNA Extraction and Analysis

Genomic DNA was extracted from regenerated shoots of tobacco, hygromycin-resistant rice calli, or regenerated rice plants, using Agencourt Chloro Pure (BECKMAN COULTER, USA), and target loci were amplified by PCR using the primer sets listed in supplemental table 1. For cleaved amplified polymorphic sequences (CAPS) analysis, PCR products were digested by the appropriate restriction enzymes, and then analyzed by agarose gel electrophoresis. A heteroduplex mobility assay (HMA) was performed using MultiNA (SHIMADZU, Japan) according to our previous report12 (link).

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Endo A., Masafumi M., Kaya H, & Toki S. (2016). Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Scientific Reports, 6, 38169.

Publication 2016

Agencourt Chloro Pure MultiNA

Agarose gel electrophoresis Assay Calli Genomic Hygromycin Mobility Polymorphic Primer Restriction enzymes Rice plants Sequences analysis Tobacco

Corresponding Organization :

Other organizations : National Agriculture and Food Research Organization, Institute of Agrobiological Sciences

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Variable analysis

independent variables

Source of genomic DNA (regenerated shoots of tobacco, hygromycin-resistant rice calli, or regenerated rice plants)

dependent variables

Amplified target loci
Restriction fragment patterns from CAPS analysis
Heteroduplex mobility patterns from HMA

control variables

Extraction method (Agencourt Chloro Pure)
PCR primer sets used to amplify target loci
Restriction enzymes used for CAPS analysis
HMA protocol (MultiNA analysis)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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