Degenerate codon mixing for PCR-based manipulation of highly repetitive sequences
Repeat 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.
We 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.
KeywordsPolyglutamine repeats Codon redundancy Degeneracy of the genetic code PCR based repeat region amplification Aggregate proteins Autophagy
80 glutamine repeats
green fluorescent protein
hours post fertilisation
polymerase chain reaction
The aberrant expansion of unstable CAG repeats coding for polyglutamine (polyQ) tracts underlies a group of neurodegenerative diseases, including Huntington’s disease (HD) and several forms of spino-cerebellar ataxia (SCA) . These diseases exhibit polyQ length-dependent toxicity, whereby age at disease onset is inversely correlated to the number of polyQ repeats . They display common cellular and molecular mechanisms including protein aggregation and inclusion body formation . Such protein aggregates depend strongly on autophagy for their clearance and dysfunction of this pathway may contribute to the pathology of these diseases . Enhancement of autophagy has been suggested to have possible therapeutic value in such diseases showing protein aggregation by promoting the clearance of these aggregates and protecting cells against their toxic effects [5, 6]. However, studying the influence of polyQ tract length on aggregation kinetics is challenging due to difficulties faced when cloning repetitive DNA sequences primarily due to the lack of unique primer binding sites [7, 8]. Previously, several polymerase chain reaction (PCR) based methods to amplify repetitive DNA regions have been described [9, 10, 11]. However, most of these either generate nonspecific products, flawed repeats, or a collection of clones with varying numbers of repeats making the identification and isolation of the specific clone of interest laborious [12, 13, 14]. In order to investigate the autophagic degradation activity or ‘autophagic flux’ of polyQ protein aggregates we sought to clone reporter constructs containing more closely defined numbers of glutamine residues. We designed a polyQ sequence with low repeat similarity by exploiting the codon redundancy of the genetic code. This strategy allowed us to amplify close to the desired numbers of glutamine repeats (although still with some variability due to two distinct causes), which we subsequently used to assess in vivo variations in ‘autophagic flux’ in a larval zebrafish model for Alzheimer’s disease .
Results and discussion
We designed a polyQ sequence containing 80 glutamine repeats (Q80). The sequence was designed to have low repeat similarity by randomly interspacing glutamine-coding CAG triplets with glutamine-coding CAA triplets (Fig. 1b, c). The nucleotide substitutions were made by eye to generate a semi random pattern. This non-repetitive sequence design should not only enhance sequence stability during propagation in bacteria but also enabled the design of PCR primers that annealed to specific regions of the sequence.
The Q80-GFP-v2A-GFP construct described above was commercially synthesised (Biomatik Corporation) (see Additional file 1) and sub-cloned via the BamHI and ClaI restriction sites into the Tol2 transposon-based, pT2AL200R150G gene transfer vector (hereafter referred to as Tol2) available from the Kawakami laboratory  (Fig. 1a and see Additional file 2).
In conclusion this study provides a robust and easily adoptable solution to generate close to intended lengths of polyQ repeats. In order to generate exact numbers of glutamine repeats subsequent rounds of amplifications with altered primer sequences can be carried out. In addition the primer length can be increased to enhance specificity of primer annealing to the template. Our technique has several advantages over the existing methods for PCR-based amplification of repetitive regions and aims to minimise the generation of non-specific PCR products and flawed repeats by exploiting the codon redundancy of the genetic code to generate a synonymous DNA coding sequence with reduced repetition. In addition, our approach is not limited to generating polyQ repeat sequences but can also be generalised to generate other nucleotide repeat sequences. Furthermore, our method is relatively cheap, as only the initial polyQ80-GFP-v2A-GFP construct requires commercial synthesis, the cost of which depends on the price per nucleotide base, length, purity and mass. All other materials required are standard reagents used for molecular cloning. In summary the technique described provides an easy to adopt, affordable solution to generate repeat-coding DNA sequences that can be manipulated as required.
Initial construct requires commercial synthesis.
May not generate the exact number of glutamine repeats required.
DR performed the experimental work and drafted this manuscript. MN supervised the laboratory work and trained DR in the necessary techniques. ML conceived the project and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its additional information files.
Consent for publication
Ethics approval and consent to participate
Conducted under the auspices of the University of Adelaide Animal Ethics Committee under permit S-2014-108.
This work was supported by a Grant from Australia’s National Health and Medical Research Council, GNT1061006.
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- 9.Riet J, Ramos L, Lewis RV, Marins L. Improving the PCR protocol to amplify a repetitive DNA sequence. Genet Mol Res. 2017;16(3). https://doi.org/10.4238/gmr16039796.
- 15.Jiang H, Newman M, Ratnayake D, Lardelli M. Ratiometric assays of autophagic flux in zebrafish for analysis of familial Alzheimer’s disease-like mutations. bioRxiv. 2018. https://doi.org/10.1101/272351.
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