TY - JOUR
T1 - Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli
AU - Godiska, Ronald
AU - Mead, David
AU - Dhodda, Vinay
AU - Wu, Chengcang
AU - Hochstein, Rebecca
AU - Karsi, Attila
AU - Usdin, Karen
AU - Entezam, Ali
AU - Ravin, Nikolai
PY - 2009/12/29
Y1 - 2009/12/29
N2 - Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.
AB - Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed 'pJAZZ' vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT-rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.
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U2 - 10.1093/nar/gkp1181
DO - 10.1093/nar/gkp1181
M3 - Article
C2 - 20040575
AN - SCOPUS:77951284583
SN - 0305-1048
VL - 38
SP - e88.1-e88.9
JO - Nucleic acids research
JF - Nucleic acids research
IS - 6
M1 - gkp1181
ER -