PCR-based cloning of the complete mouse mitochondrial genome and stable engineering in Escherichia coli.
We have devised a method for cloning an entire mammalian mitochondrial genome (mtDNA) in Escherichia coli using PCR-based amplification and sequential ligation. Here we test this approach by cloning the complete mouse mtDNA. The mtDNA was divided into four to five fragments based on unique restriction enzyme sites and amplified by high-fidelity long-range DNA polymerase. The synthesized fragments were cloned individually to test their toxicity in the E. coli host and then combined sequentially into a vector containing the E. coli R6K origin of DNA replication. The synthetic complete mouse mtDNA clones were replicated stably and faithfully in E. coli when maintained at moderately low copy numbers per cell. The sequence integrity of the synthetic mouse mtDNA clones was confirmed by nucleotide sequencing; no mutations or rearrangements in the genome were found. This approach can facilitate the cloning of entire mammalian mitochondrial genomes in E. coli and assist in the introduction of desired modifications into the mitochondrial genome.
Yoon
Young Geol
YG
Department of Laboratory Medicine and Pathology, Institute of Human Genetics, University of Minnesota Medical School, 420 Delaware St. S.E., Minneapolis, MN 55455, USA. yoonx015@umn.edu
Yang
Yi-Wei
YW
Koob
Michael D
MD
D000818:Animals; D002999:Clone Cells; D003001:Cloning, Molecular; D004272:DNA, Mitochondrial / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D054629:Genome, Mitochondrial / Q000235:genetics*; D051379:Mice / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004272:DNA, Mitochondrial