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Synthetic Gene Construction (Madan) Library Method Overview

To target missing genes from the MGC, a high throughput PCR-based recombinant method was used in the laboratory of Anup Madan, University of Iowa. Primers were designed with specific extensions for each exon in a gene, which permitted neighboring exons to hybridize to one another, thus forming an overlap. Extension of this overlap by DNA polymerase then yielded a recombinant molecule. This process is referred to as gene Splicing by Overlap Extension (SOE) or gene SOEing.

Note: In practice, more than one gene was prepared at a time but the method below provides a simplified overview for one gene with four exons.
  • Exon and BAC Selection
    The sequence of the gene to be synthesized was first searched against the genomic sequence using the UCSC Genome browser to identify:
    1. The start and stop position of exons
    2. The BAC(s) to be used as templates for amplification of these exons.

  • PCR Primer Design
    1. The primers b, d, and f were designed with 20nt overhangs on their 5’ ends that were complementary to the 5’ ends of the exon to their immediate right. Primers c, g, and e had overhangs of 20nt at their 3’ ends which were complementary to the 3’ end of the exon immediate left. If the size of the exon was smaller than 40 bp, it was included in the primers for neighboring exons.

  • Template Preparation
    1. The BAC(s) that spanned the gene was picked from the BAC libraries and streaked onto LB-agar plates with appropriate antibiotics.
    2. A single colony was inoculated in liquid media and grown for 18 hours. BAC DNA was extracted using an AutoGenprep740.
    3. The DNA was digested with EcoRI enzyme and resolved on 1.0% agarose gel to check the quality of the BAC DNA. If the BAC DNA did not meet the quality standards it was prepared again. After quality was assured, BAC DNA was dispensed into 96 well plates for long term storage.

  • Gene Assembly-PCR and SOE Reactions
    1. Exons were amplified by PCR in 96-well plates using high fidelity Platinum Taq polymerase (Invitrogen). (PCR1, 2, 3, and 4)
    2. The PCR products were analyzed by agarose gel electrophoresis. The size of the observed band was compared with the predicted size of the SOE product.
    3. Products from PCR1 and 2 were combined, as were products from PCR3 and 4. The separate mixtures were dissociated and annealed (SOE1 and SOE2).
    4. The two products of SOE1 and SOE2 were amplified using high-fidelity Platinum Taq polymerase from Invitrogen and reaction products were analyzed by agarose gel electrophoresis. The process was repeated iteratively until the final product was obtained.

  • Cloning and Sequence Verification
    1. Final SOE constructs were purified using with the QIAquick® 96-well PCR purification kit (Qiagen)
    2. The fragments were cloned into either pUC19 or pCR Blunt II Topo (Invitrogen) vector. The resulting plasmid DNA was prepared and sequenced with universal forward and reverse primers with BIG Dye Terminator Version 3.1.
    3. These insert sequences were checked against the RefSeq record of the gene. Those clones with correct 3’ and 5’ ends were sequenced further to 1 error per 50,000 bp accuracy.