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Efficient identification of TALEN ‐mediated genome modifications using heteroduplex mobility assays
Author(s) -
Ota Satoshi,
Hisano Yu,
Muraki Michiko,
Hoshijima Kazuyuki,
Dahlem Timothy J.,
Grunwald David J.,
Okada Yasushi,
Kawahara Atsuo
Publication year - 2013
Publication title -
genes to cells
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.912
H-Index - 115
eISSN - 1365-2443
pISSN - 1356-9597
DOI - 10.1111/gtc.12050
Subject(s) - transcription activator like effector nuclease , heteroduplex , biology , indel , amplicon , genetics , microbiology and biotechnology , electrophoretic mobility shift assay , genome , dna , computational biology , gene , polymerase chain reaction , genome editing , transcription factor , single nucleotide polymorphism , genotype
The heteroduplex mobility assay ( HMA ) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1–10 bp deletions) in the multiple cloning site ( MCS ) of p B luescript II. After PCR amplification of the MCS using a mixture of wild‐type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator‐like effector nucleases ( TALEN )‐induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN ‐injected F0 embryos. Furthermore, TALEN ‐injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN ‐mediated genome modifications.