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Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.

ABSTRACT: The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three different enzymes--T7 endonuclease I, T4 endonuclease VII and Escherichia coli endonuclease V--have been tested. As a model, a synthetic polynucleotide encoding the bacterial chloramphenicol-acetyltransferase (cat) was synthesized using different methods for one step polynucleotide synthesis based on ligation of oligonucleotides. The influence of enzymatic mismatch cleavage (EMC) as an error correction step on the frequency of correct products was analyzed by functional cloning of the synthetic cat and comparing the error rate with that of untreated products. Significant reduction of all mutation types was observed. Statistical analysis revealed that the T4 and E.coli endonucleases reduced the occurrence of mutations in cloned synthetic gene products. The EMC treatment was successful especially in the removal of deletions and insertions from the primary ligation products.

SUBMITTER: Fuhrmann M

PROVIDER: S-EPMC1072809 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.

Fuhrmann Markus M Oertel Wolfgang W Berthold Peter P Hegemann Peter P

Nucleic acids research 20050330 6

The success of long polynucleotide de novo synthesis is largely dependent on the quality and purity of the oligonucleotides used. Generally, the primary product of any synthesis reaction is directly cloned, and clones with correct products have to be identified. In this study, a novel strategy has been established for removing undesired sequence variants from primary gene synthesis products. Single base-pair mismatches, insertions and deletions were cleaved with specific endonucleases. Three dif ...[more]

PMID: 15800209

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Project description:BackgroundDoubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited.ResultsWe have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers.ConclusionsEnzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines.

Dataset Information

Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.

Publications

Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage.

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