Overcoming a barrier for DNA polymerization in triplex-forming sequences

Abstract

Folded structures in the DNA template, such as hairpins and multi-stranded structures, often serve as pause and arrest sites for DNA polymerases. DNA polymerization is particularly difficult on mirrorrepeated homopurine·homopyrimidine templates where triple-stranded (triplex) structures may form between the nascent and folded template strands. In order to use a linear PCR amplification approach for the structural analysis of DNA in mirror-repeated sequences we modified a conventional protocol. The barrier for DNA synthesis can be eliminated using an oligonucleotide that hybridizes with the template to prevent its folding and is subsequently displaced by the progressing polymerase. The described approach is potentially useful for sequencing and analysis of chemical adducts and point mutations in a variety of sequences prone to the formation of folded structures, such as long hairpins and quadruplexes.

Disruption of Higher Order DNA Structures in Friedreich’s Ataxia (GAA)n Repeats by PNA or LNA Targeting

“…As controls for the PE reactions, plasmids were incubated under similar conditions in the absence of BQQ-OP or CAA. Sequencing ladders were prepared by using plasmids (100 ng) that had been cleaved by using PstI and SacI as templates for the PE reactions in the presence of dideoxynucleotides as described in the literature [59]. All samples were analyzed using denaturing polyacrylamide gel electrophoresis (6%, 7 M urea, 0.5 mm) in buffer (1X TBE) at room temperature and 1200 V, 32 mA, 2.5 h. Fuji FLA3000 phosphorimager was used for scanning, analysis and gel bands quantification.…”

Slipped strand DNA structures

“…Py•Pu tracts with mirror repeat symmetry have long been known to block the DNA replication fork in cells (92,93). Replication fork blockage may result from the folding of a single strand DNA template back into the major groove of the nascent duplex in part of the Py•Pu tract, resulting in the formation of triplex DNA (93)(94)(95). Strong blocks to DNA replication have also been observed when triplex structures are formed prior to polymerization (96).…”

“…(GAA) n •(TTC) n repeats can also form triplex DNA (29, [97][98][99][100][101][102] and pause DNA replication in vitro within the repeat tract (101), as observed for other triplex-forming DNA sequences (93)(94)(95). The formation of a [(GAA) n •(dGAA) n •(dTTC) n ] RNA-DNA triplex during transcription of the (GAA) n •(TTC) n repeat in the frataxin gene has also been proposed as a mechanism of gene downregulation.…”

3′ RACE LaNe: a simple and rapid fully nested PCR method to determine 3′-terminal cDNA sequence

“…However, despite their capacity for the suppression of nonspecific PCR products (4), they remain essentially one-sided reactions. As such, they can be compromised in their selection of specific products in difficult scenarios, such as long coding or 3 untranslated regions and regions including high GC content, secondary structure, or homopolymeric tracts (5,6). …”