Inhibition of DNA synthesis by K⁺‐stabilised G‐quadruplex promotes allelic preferential amplification

Abstract

PCR preferential amplification consists of the inefficient amplification of one allele in a heterozygous sample. Here, we report the isolation of a GC-rich human minisatellite, MsH43, that undergoes allelic preferential amplification during PCR. This effect requires the existence of a (TGGGGC) 4 motif that is able to form a G-quadruplex in the presence of K þ . This structure interferes with the DNA synthesis of the alleles harbouring this motif during PCR The present results are the first demonstration that the formation of G-quadruplex can be one of the mechanisms involved in some kinds of preferential amplification.

G4 Resolvase 1 Binds Both DNA and RNA Tetramolecular Quadruplex with High Affinity and Is the Major Source of Tetramolecular Quadruplex G4-DNA and G4-RNA Resolving Activity in HeLa Cell Lysates

“…These sequences form DNA or RNA quadruplexes under physiological salt conditions and have high melting temperatures that human cells could not endure. Quadruplex structures are expected to form in cells under physiological conditions at least in a stochastic manner and these bulky complexes have been shown in vitro to impede normal DNA and RNA metabolism (25,34). Thus, an essential cellular problem emerges with quadruplex formation in vivo impeding normal metabolism and it invites an enzymatic solution by specific G4-nucleic acid resolvases.…”

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

“…Signals were clearly generated for each of the four DNA templates, resulting in about equal intensities except for the G 20 template sequence, which generated a moderately smaller signal, possibly due to Gquadruplex structures that may inhibit the polymerase. 18 In all four cases, signals were considerably (13-33-fold) higher than background lacking primer/template.Next we tested sequence selectivity of the chimeric nucleotides, evaluating sixteen combinations of ARNs with the four DNA sequences ( Figure 2C and Figure S1). In all cases, the correct nucleotide/target sequence combinations yielded much higher signals than incorrect combinations, showing clear nucleotide/template base selectivity.…”

“…Signals were clearly generated for each of the four DNA templates, resulting in about equal intensities except for the G 20 template sequence, which generated a moderately smaller signal, possibly due to Gquadruplex structures that may inhibit the polymerase. 18 In all four cases, signals were considerably (13-33-fold) higher than background lacking primer/template.…”

Heteroduplex analysis of minisatellite variability

“…In other cases, the substrate of PCR was 0.1 ng of recombinant plasmid DNA containing MsH43 alleles [22]. Reactions were performed in a volume of 25 mL containing PCR buffer (67 mM Tris-HCl, pH 8.8, 16 mM (NH 4 ) 2 SO 4 , 0.01% Tween-20), 0.3 mM of each primer P02.1 (5'-GAATTCTATCTTTCCTGCAAAC-3'), and P02.2 (5'-TATACCCAGTGACCAAGGCC-3'), 0.2 mM dNTPs, 1.5 mM MgCl 2 , and 0.5 U of Taq polymerase (Genecraft).…”