Generation of Long Tracts of Disease-Associated DNA Repeats

Abstract

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.

A Z-DNA sequence reduces slipped-strand structure formation in the myotonic dystrophy type 2 (CCTG)·(CAGG) repeat

“…For plasmids containing (CCTG)⅐(CAGG) repeats and human flanking sequences, the DNA sequences were cut from plasmids containing PCR products of human alleles. For plasmids lacking human flanking sequences, repeats were generated as described, adding EcoRI linkers such that the (CCTG)⅐(CAGG) repeat is flanked by EcoRI sites (48).…”

Directed PCR-free engineering of highly repetitive DNA sequences

“…Our approach is also suitable to increase the length of repetitive sequences by multiple insertions as described previously [13]. Thereby, simultaneous digestion of the annealed oligonucleotides or pMK1-Q n plasmids with both Type IIS restriction enzymes would result in Q-block fragments with compatible overhangs (Figure 1A).…”

“…However, most of them include PCR-based amplification steps, generate imperfect repeats, or result in a pool of clones that differ in the number of repeats [6-13]. As a consequence, additional effort is required to identify and isolate clones with the desired length of nucleotide repeats.…”

Cell-free cloning of highly expanded CTG repeats by amplification of dimerized expanded repeats

“…In the case of CTG repeats, despite optimization of host and vector combinations, the upper limit that can be maintained is generally <300, owing to the propensity of E. coli to delete repeat tracts [(10), however, see reference (11) for an exception]. In vitro ligation of pure CTG repeats is also inefficient owing to strand slippage that changes the overhangs at the ends of the DNA molecules (12). Despite recent technical advances that yield greater ligation efficiencies, it is still difficult to generate ligation products with >300 CTG repeats (12).…”

“…In vitro ligation of pure CTG repeats is also inefficient owing to strand slippage that changes the overhangs at the ends of the DNA molecules (12). Despite recent technical advances that yield greater ligation efficiencies, it is still difficult to generate ligation products with >300 CTG repeats (12). …”