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pCR4-TOPO/pCR-XL-TOPO 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 Dr. Marco Marra. The original method is described in detail in "Systematic Recovery and Analysis of Full-ORF Human cDNA Clones (2004)": it used the pCR4-TOPO vector (Invitrogen) and the Advantage-HF 2 Polymerase (Clontech) for the PCR step. However, the vector was subsequently changed to pCR-XL-TOPO (Invitrogen) to allow insertion of amplicons >4kb. In addition, the PCR enzyme was changed to Phusion™ polymerase (MJ Research), a high fidelity DNA polymerase.

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 or total RNA was obtained from Dr. Michael Brownstein (NIH). In this latter case, mRNA was isolated using µMACS Oligo(dT) MicroBeads from Miltenyi Biotec.
    • The mRNA was converted to double stranded cDNA using the SuperScript Choice System for cDNA Synthesis (Invitrogen). Briefly:
      • First strand cDNA was synthesized using oligo-(dT)12-18 primers and SuperScriptTM II or III reverse transcriptase, depending on the target's length.
      • Second strand cDNA was synthesized using E. coli DNA polymerase I in conjunction with E. coli RNase H and E. coli DNA ligase.
    • The cDNA was extracted by phenol:chloroform (1:1), ethanol precipitated, and resuspended in TE buffer.
  • PCR Primer Design
    • The sequences of targeted genes were downloaded from GenBank. Each contained a putative complete ORF, though not necessarily the untranslated region (UTR) sequences.
    • Gene specific primer pairs were designed, based on the following criteria:
      • Optimal Tm 60oC, minimum Tm 55oC, and maximum Tm 67oC.
      • Size range between 18 and 27nt
      • GC content between 20% and 80%
      • Maximum allowable local alignment scores for self-complementary and 3’ self-complementary, 8 and 3 respectively
      • No further than 100 bp from the ends of the ORF
  • PCR
    • The cDNAs were amplified by PCR in 96-well plates using Advantage-HF 2 Polymerase (Clontech), which was subsequently changed to Phusion™ polymerase (MJ Research)(see Table below).
    Advantage-HF 2 Polymerase LibrariesPhusion™ Polymerase Libraries
    NIH_MGC_245
    NIH_MGC_264 to NIH_MGC_268
    NIH_MGC_271 to NIH_MGC_276
    NIH_MGC_286 to NIH_MGC_329
  • Cloning
    • The PCR product was separated on a 1% agarose gel, visualized with SYBR Green (Mandel), excised, and purified with either MinElute or Qiaquick gel extraction kits for amplicons <4kb or >4kb, respectively. (Qiagen).
    • Purified amplicons <4kb were cloned into the pCR4-TOPO vector using the TOPO TA Cloning kit from Invitrogen. Purified amplicons >4kb were cloned into the pCR-XL-TOPO vector using the pCR-XL-TOPO PCR cloning kit from Invitrogen.