12 Life Technologies
™
| Parkinson's cell models
iPSC colony screening methods
The GeneArt
®
Genomic Cleavage Detection Kit was used
to detect indels generated by the GeneArt
®
Precision TALs
pair, or to detect "corrections" of mutant sequences by the
introduction of wild type sequences at the desired loci. The
assay is shown schematically in Figure 3. While this method
is sensitive for detecting mutations that are introduced
and that lead to formation of a heterozygous mutation, it
can be difficult to detect a subtle sequence change from
a heterozygous to homozygous allele, especially when the
event is present in only in a small subset of cells in a colony.
GeneArt
®
Genomic Cleavage Detection assay
Indel
Mismatch
PCR amplification (no purification)
1
Denaturation and re-annealing
2
Mismatch detection and cleavage
3
Agarose gel electrophoresis
4
Indel
Mismatch
PCR amplification (no purification)
1
Denaturation and re-annealing
2
Mismatch detection and cleavage
3
Agarose gel electrophoresis
4
Figure 3. GeneArt
®
Genomic Cleavage Detection assay. To detect either an indel or a
mutation within a specific sequence of DNA, the region is first amplified using primers
specific for that region. A second nested PCR can be performed to increase sensitivity.
After heating the sample and re-annealing the PCR products, amplicons containing indels
or other changes in sequence will result in the formation of heteroduplexes with amplicons
containing non-modified sequences. When these heteroduplexes are treated with an
endonuclease that only cleaves in the presence of a mismatch, two pieces of DNA of known
size are generated, which can be detected by agarose gel electrophoresis.
