PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications.

Designer transcription-activator like effectors (TALEs) is a promising technology and made it possible to edit genomes with higher specificity. Such specific engineering and gene regulation technologies are also being developed using RNA-binding proteins like PUFs and PPRs. The main feature of TALEs, PUFs and PPRs is their repetitive DNA/RNA-binding domains which have single nucleotide binding specificity. Available kits today allow researchers to assemble these repetitive domains in any combination they desire when generating TALEs for gene targeting and editing. However, PCR amplifications of such repetitive DNAs are highly problematic as these mostly fail, generating undesired artifact products or deletions. Here we describe the molecular mechanisms leading to these artifacts. We tested our models also in plasmid templates containing one copy versus two copies of GFP-coding sequence arranged as either direct or inverted repeats. Some limited solutions in amplifying repetitive DNA regions are described.

Reevaluation of the reliability and usefulness of the somatic homologous recombination reporter lines.

A widely used approach for assessing genome instability in plants makes use of somatic homologous recombination (SHR) reporter lines. Here, we review the published characteristics and uses of SHR lines. We found a lack of detailed information on these lines and a lack of sufficient evidence that they report only homologous recombination. We postulate that instead of SHR, these lines might be reporting a number of alternative stress-induced stochastic events known to occur at transcriptional, posttranscriptional, and posttranslational levels. We conclude that the reliability and usefulness of the somatic homologous recombination reporter lines requires revision. Thus, more detailed information about these reporter lines is needed before they can be used with confidence to measure genome instability, including the complete sequences of SHR constructs, the genomic location of reporter genes and, importantly, molecular evidence that reconstituted gene expression in these lines is indeed a result of somatic recombination.