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Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

ABSTRACT: A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.

SUBMITTER: Ciccarelli RB

PROVIDER: S-EPMC329059 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

Ciccarelli R B RB Gunyuzlu P P Huang J J Scott C C Oakes F T FT

Nucleic acids research 19911101 21

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions w ...[more]

PMID: 1945885

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Project description:Overlapping genes (OLGs) with long protein-coding overlapping sequences are disallowed by standard genome annotation programs, outside of viruses. Recently however they have been discovered in Archaea, diverse Bacteria, and Mammals. The biological factors underlying life's ability to create overlapping genes require more study, and may have important applications in understanding evolution and in biotechnology. A previous study claimed that protein domains from viruses were much better suited to forming overlaps than those from other cellular organisms - in this study we assessed this claim, in order to discover what might underlie taxonomic differences in the creation of gene overlaps. After overlapping arbitrary Pfam domain pairs and evaluating them with Hidden Markov Models we find OLG construction to be much less constrained than expected. For instance, close to 10% of the constructed sequences cannot be distinguished from typical sequences in their protein family. Most are also indistinguishable from natural protein sequences regarding identity and secondary structure. Surprisingly, contrary to a previous study, virus domains were much less suitable for designing OLGs than bacterial or eukaryotic domains were. In general, the amount of amino acid change required to force a domain to overlap is approximately equal to the variation observed within a typical domain family. The resulting high similarity between natural sequences and those altered so as to overlap is mostly due to the combination of high redundancy in the genetic code and the evolutionary exchangeability of many amino acids. Synthetic overlapping genes which closely resemble natural gene sequences, as measured by HMM profiles, are remarkably easy to construct, and most arbitrary domain pairs can be altered so as to overlap while retaining high similarity to the original sequences. Future work however will need to assess important factors not considered such as intragenic interactions which affect protein folding. While the analysis here is not sufficient to guarantee functional folding proteins, further analysis of constructed OLGs will improve our understanding of the origin of these remarkable genetic elements across life and opens up exciting possibilities for synthetic biology.

Dataset Information

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

Publications

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

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