"Development of a TA cloning- and RE digestion-based method to examine genetic rearrangements of TALE repeat arrays. ( a ) Work flow chart. A fusion expression cassette bearing both left and right TALEN arms (TALEN L-R) is amplified with forward and reverse PCR primers (FP and RP) designed to amplify the full-length of tandem repeat arrays. The PCR products are inserted into a TA cloning vector. EcoRI digestion of individual clones followed by agarose gel electrophoresis is used to detect the size change of the TALE repeat arrays. The detection of a 1.76 kb DNA fragment indicates that the number of the tandem repeat units remains correct in the TALEN expression cassette. To further classify the genetic rearrangement events, the clones with no obvious size changes are subjected to DNA sequencing. ( b ) The use of an enzyme mixture allows PCR amplification of single band. Left: Platinum Taq DNA Polymerase High Fidelity. Right: Platinum Taq DNA Polymerase High Fidelity mixed with Elongase (1:2). ( c ) TA cloning and EcoRI digestion detect no change in the size of the TALE repeat arrays in plasmid vector samples. The band at 3 kb after EcoRI digestion is the linearized cloning vector, while the DNA band at 1.76 kb is the DNA insert bearing a TALE repeat array. ""–"" and ""+"": Without and with EcoRI. One kb DNA ladder was used as a molecular weight standard. ( d ) DNA sequencing analysis confirms no change in DNA recognition specificity by RVDs in left (top panel) and right (bottom panel) TALEN arms in the above plasmid samples. Amino acid sequence alignment was performed after translating DNA sequences into amino acids. The pair letters in the sequence represent a RVD (HD, NI, NG, and NN) for each TALE repeat unit in a TALEN arm."
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