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Human Full-length Expression Clone Library Overview Method

Full–length clones for human kinases in the master vector pDNR_Dual (BD Biosciences) were generated in the laboratory of Dr. Joshua LaBaer at the Harvard Institute of Proteomics. For each gene, the complete open reading frame (ORF) was directionally cloned. Two versions of each ORF were prepared: both versions contained a start codon but one ended with a stop codon (closed version) while the other ended with a leucine instead of the stop codon to allow fusion with a C-terminal tag (fusion version).

The method by Park et al. is described in detail in PNAS 2005; 102(23):8114-9. Briefly the methods were as follows:

  • Starting materials were either:
    • Plasmid DNA isolated from MGC clones that were obtained from LLNL.
    • mRNA purified from placenta and brain tissue and converted into first strand cDNA using Invitrogen's SuperScript™ First-Strand Synthesis System for RT-PCR.
  • Primers were designed that contained both: at least 15 bases of homology with the sequence flanking the desired site of insertion of the the cloning vector joined to 20-24 bases homologous to the specific gene.
  • PCR was performed using Invitrogen's Pfx polymerase for 12 cycles if plasmid DNA was the template or for 40 cycles if cDNA first strand was the template. After November 2004, all PCR used Pfu polymerase.
  • PCR products were separated by agarose gel. Bands containing fragments of the predicted size were cut out of the gel and isolated using spin-filtration 96-well plates (initially Millipore GF/D plates, then 3µm glass fiber plates from Innovative Microplate).
  • Purified amplicons were cloned into the master vector pDNR_Dual (BD Biosciences) using the method described in the BD In-Fusion Cloning Kit User Manual, with one minor modification: the total reaction mixture was 10µl instead of 20µ;l.
  • A commercial company sequenced the entire insert as well as the 5' and 3' transitions between the vector and the gene. Discrepancies if present were annotated in the released gene/clone sequence. Clones were not accepted if amino acid changes greater than 1 were present except for clones from cDNA first strand if the additional changes were previously documented variations for the gene (isoform or polymorphism).