Directed PCR-free engineering of highly repetitive DNA sequences.

Annika Scior,Steffen Preissler,Miriam Koch,Elke Deuerling

doi:10.1186/1472-6750-11-87

Abstract

BackgroundHighly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products.ResultsFor the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays.ConclusionOur seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

Highlights

Repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome
We designed double-stranded oligonucleotides containing a defined number of central glutamine-encoding CAG and CAA triplets (Q-block) arranged in a non-regular fashion (Figure 1A). We decided on this approach because it has been shown previously that interruption of perfect CAG repeats by CAA triplets improves the stability of Poly-Q encoding sequences in E. coli [12]
The central triplet repeats were flanked by non-repetitive sequences that have two important functions: First, they ensure the specific annealing of the oligonucleotides, and secondly, they comprise two inward directed Type IIS restriction sites (BsaI and BsmBI) and a Type IIP restriction site (SacI)

Summary

Introduction

Repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Studying repetitive sequences is of biological, biotechnological and medical relevance. Cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. One of the best-studied examples for a trinucleotide expansion disease is the neurological disorder Huntington’s chorea, where the accumulation of CAG triplets within the first exon of the gene encoding the Huntingtin (Htt) protein leads to an elongated polyglutamine (Poly-Q) stretch in the polypeptide. We can recombine constructs of the same or different repeat lengths to accelerate the construction of the desired number of repeats

Methods

Results

Conclusion