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.
Plant Molecular Engineering Group, IZMB (Institute of Cellular and Molecular Botany), University of Bonn, Kirschallee 1, 53115 Bonn.
Carl Maximilian
CM
Hommelsheim
1] Plant Molecular Engineering Group, IZMB (Institute of Cellular and Molecular Botany), University of Bonn, Kirschallee 1, 53115 Bonn [2].
Lamprinos
L
Frantzeskakis
Plant Molecular Engineering Group, IZMB (Institute of Cellular and Molecular Botany), University of Bonn, Kirschallee 1, 53115 Bonn.
Mengmeng
M
Huang
Plant Molecular Engineering Group, IZMB (Institute of Cellular and Molecular Botany), University of Bonn, Kirschallee 1, 53115 Bonn.
Bekir
B
Ülker
D003851:Deoxyribonucleases / Q000378:metabolism; D018390:Gene Targeting; D005818:Genetic Engineering; D015894:Genome, Human; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*
D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases