Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences

Abstract

ObjectiveRepeat expansion of polyglutamine tracks leads to a group of inherited human neurodegenerative disorders. Studying such repetitive sequences is required to gain insight into the pathophysiology of these diseases. PCR-based manipulation of repetitive sequences, however, is challenging due to the absence of unique primer binding sites or the generation of non-specific products.ResultsWe have utilised the degeneracy of the genetic code to generate a polyglutamine sequence with low repeat similarity. This strategy allowed us to use conventional PCR to generate multiple constructs with approximately defined numbers of glutamine repeats. We then used these constructs to measure the in vivo variation in autophagic degradation activity related to the different numbers of glutamine repeats, providing an example of their applicability to study repeat expansion diseases. Our simple and easily generalised method of generating low repetition DNA sequences coding for uniform stretches of amino acid residues provides a strategy for generating particular lengths of polyglutamine tracts using standard PCR and cloning protocols.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3298-5) contains supplementary material, which is available to authorized users.

Rapid and Accurate Screening of Lysine-Producing Edible Mushrooms via the Homocitrate Synthase Gene as a Universal Molecular Marker

“…Genomic DNA was extracted from mushroom mycelial tissues following the modified sodium dodecyl sulfate method. 26 Among the different strains, 73 strains were developed under the same culture conditions, consistent with that reported by Kala et al 4 (Table 1). The strains were cultured for genomic DNA extraction at 28 °C for 5−7 days.…”