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pPCR-Script™ Amp SK(+) Library Method Overview

A scalable method for targeted generation of cDNA clones to facilitate recovery of genes absent from the MGC collection was developed in the laboratory of Drs. Richard Gibbs and John McPherson. The method is described in detail in "Large-scale RT-PCR recovery of full-length cDNA clones (2004)" and is outlined below:

Note: This protocol is in the process of being optimized to improve clone recovery.
  • cDNA Synthesis
    • mRNA from various human tissues was purchased from commercial companies.
    • First stranded cDNA was synthesized using oligo-(dT)12-18 and SuperScript II Reverse Transcriptase (Invitrogen). Pfu and 10% DMSO were included in the reaction to enhance the synthesis of longer products.
    • mRNA was cleaved with RNase H and aliquots of the final mixture were used in the PCR.

  • PCR Primer Design
    • The sequence of the targeted gene was downloaded from GenBank. It contained a putative complete ORF, though not necessarily the untranslated region (UTR).
    • Algorithms were developed to pick primers within the 5’ and 3’ UTR. If UTR information was not available, primers were positioned precisely at the beginning and end of the ORF.
    • Gene specific primer pairs were designed based on "Restricted" criteria, the most stringent and, theoretically, the best. If these criteria did not result in primers, "Relaxed" criteria were used, and finally "Forced". The Restricted Criteria were:
      • Complete ORF
      • Starting primer position 80 bp or more from ORF
      • Primer length between 25 and 30 bp
      • GC content between 35% and 75%
      • Tm range between 65oC and 80oC
      • Avoided hair pin structure of three base pairs or more
      • Avoided homology with other sequences
      • Avoided homopolymeric runs of three base pairs or more
      • Avoided GC clamps

  • PCR
    • The cDNA first strand was amplified by PCR in 96-well plates using AdvantageTM 2 Enzyme System (BD Biosciences Clontech.)

  • Cloning
    • PCR products were purified using with the QIAquick® 96-well PCR purification kit (Qiagen).
    • Purified amplicons were cloned into the pPCR-Script™ Amp SK(+) vector utilizing the PCR-Script® Amp, Cloning Kit (Stratagene).