Supplementary Figure 3: Comparison with other methods. | Nature Protocols

Supplementary Figure 3: Comparison with other methods.

From: Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification

Supplementary Figure 3

a) Left panel: Results obtained by Ligation-Mediated Purification (this experiment is also shown in Figure 3b). Right panel: Extent of cleavage in the same samples measured by qPCR with primers on each side of the break (5'-CACTATAGGGAGACCCAAGCTG and 5'-CTGGTCGGCGATTAGCGACT). Errors bars represent standard deviation of the qPCR measurements. The % of cleavage is given by the decrease in PCR efficiency (represented by the arrow). Note that the variations between two identical samples (the +I-SceI sample and the no-ligase sample which should give the same signal since they are produced from the same genomic DNA) are higher than variations between different genomic DNA. Thus, the decrease observed in the +I-SceI sample is not significant, and cleavage efficiency in this experiment is below the detection level of this method. b) Left panel: Results obtained by Ligation-Mediated Purification as shown in Figure 3c, but with a log scale. Right panel: a similar standard curve of I-SceI-digested versus uncleaved genomic DNA was analysed by LM-PCR as described in 30, 31 except that genomic DNA was treated by T4 DNA polymerase following I-SceI-mediated digestion to obtain blunt ends. Quantification of ligation products was performed by qPCR using the following primers: 5'-GGGAGATCTGAATTCCCCTA and 5'-GTCGGCGATTAGCGACTTCT. Note that the sensitivity of ligation-mediated purification is about 10-fold higher than that of LMPCR.

Back to article page