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Synthetic Gene Construction (Bhat) Library Method Overview

To target missing genes from the MGC, a PCR-based recombinant method was used in the laboratory of Narayan Bhat, SAIC Frederick Inc., NCI Frederick. Gene specific primers designed to encompass the entire ORF were used to amplify cDNA.
  • cDNA Synthesis
    mRNA was obtained from various human cell lines used for MGC cDNA library construction, from human tissue prepared through collaboration with Dr. Mark A. Watson, Washington University School of Medicine, St. Louis, MO., or purchased from BD Biosciences.
    • First strand cDNA was synthesized from mRNA using reverse transcriptase SuperScript III (Invitrogen), with either the oligo-dT primer supplied with the kit or a 3’ end gene specific primer, following the manufacture’s protocol. At the end of the reaction, reverse transcriptase was inactivated by heating at 85oC for 15 minutes and used directly for RT- PCR.
  • PCR Primer Design
    • The full length Open Reading Frame (ORF) sequence for the gene of interest was obtained from the RefSeq database.
    • Gene specific primer pairs were designed using the Primer Pick 3 Program and were within the 5’ and 3’ untranslated regions, close to the ORF.
    • If there was not enough sequence information available from 5’ and 3’ untranslated regions, primers were chosen from the first and last 20 nucleotides encompassing the start and stop codons, respectively.
    • Most of the gene specific primers selected were of 20nt in length, with 50% GC content (excluding forward and reverse infusion primers) and had a Tm of ~ 60oC.
  • PCR
    The cDNAs were amplified by PCR in 96-well plates using the Advantage-HF 2 Polymerase kit from Clontech. Typical PCR conditions were:
    • 94oC for 2min
    • 30 cycles of 94oC for 30sec, 65oC for 30sec, and 72oC per min for every 1000bp
    • Final extension at 72oC for 7 min
  • Cloning
    • PCR products were analyzed on an Agilent 2100 Bioanalyzer.
      • If the size of the PCR product was >70% of the ORF, it was purified using a commercial cartridge (Qiagen, Invitrogen or BD Biosciences)
      • If the PCR product contained contaminating DNA, the ORF segment was isolated using the S.N.A.P.™ UV-Free Gel Purification Kit from Invitrogen.
    • PCR products were separated on agarose gel. ORF size DNA fragments were cut from the gel and DNA was isolated using Free Gel Purification Kit from Invitrogen following the manufacturer’s protocol.
    • The ORF was then cloned into the pDNR-Dual vector using the BD In-Fusion™ Dry-Down PCR Cloning Kit, following the manufacturer’s method.
    • Colonies were screened for full inserts by sequencing the 5' and 3' ends: up to 10 clones per gene were sequenced at both ends if the target was < 2 kbp, and up to 15 clones per gene if the target was > 4kbp.
    • Clones containing putative full ORFs were sent for full length sequencing: up to 5 clones per gene for target ORFs <2 kbp and up to 10 clones per gene for target ORFs >4 kbp.