PCR Amplification of GC-Rich DNA Regions Using the Nucleotide Analog N ⁴ -Methyl-2′-Deoxycytidine 5′-Triphosphate

Abstract

GC-rich DNA regions were PCR-amplified with Taq DNA polymerase using either the canonical set of deoxynucleoside triphosphates or mixtures in which the dCTP had been partially or completely replaced by its N4-methylated analog, N4-methyl-2'-deoxycytidine 5'-triphosphate (N4me-dCTP). In the case of a particularly GC-rich region (78.9% GC), the PCR mixtures containing N4me-dCTP produced the expected amplicon in high yield, while mixtures containing the canonical set of nucleotides produced numerous alternative amplicons. For another GC-rich DNA region (80.6% GC), the target amplicon was only generated by re-amplifying a gel-purified sample of the original amplicon with N4me-dCTP-containing PCR mixtures. In a direct PCR comparison on a highly GC-rich template, mixtures containing N4me-dCTP clearly performed better than did solutions containing the canonical set of nucleotides mixed with various organic additives (DMSO, betaine, or ethylene glycol) that have been reported to resolve or alleviate problems caused by secondary structures in the DNA. This nucleotide analog was also tested in PCR amplification of DNA regions with intermediate GC content, producing the expected amplicon in each case with a melting temperature (Tm) clearly below the Tm of the same amplicon synthesized exclusively with the canonical bases.

Testing the effect of pollen exine rupture on metabarcoding with Illumina sequencing

“…The only consistent result we observed across all barcodes and primer pairs was the nearly complete exclusion of Zea mays. This could be due to the high GC content of this species, DNA regions with a GC content in excess of 60% require special PCR protocols to overcome strong secondary structures that interfere with primer annealing [50]. We augmented our PCR protocol with the addition of 5% DMSO, and were successful with amplification of Z. mays when no other species were contained in the sample, but this addition does not seem to be sufficient for returning this species from a mixed species sample.…”

N 4-acyl-2′-deoxycytidine-5′-triphosphates for the enzymatic synthesis of modified DNA

“…Chemical synthesis, thermal stabilities and hybridization capabilities of ONs containing various cytidine nucleotides with N 4 -acyl, N 4 -alkoxycarbonyl and N 4 -carbamoyl residues have been previously described ( 45 ). Moreover, it is known that N 4 -acetyl-CTP is efficiently used as a substrate in a T7 RNA polymerase-catalysed in vitro transcription ( 46 ), whilst N 4 -alkyl-deoxycytidines have been tested for polymerase chain reaction (PCR) amplification of GC-rich DNA regions ( 47 ). Altogether, it is obvious that by gaining more details about this type of modification as well as by screening for polymerases that would use such nucleotides, it would be possible to deepen our understanding of a biological role of N 4 -modified-cytidine.…”

MS4A2-rs573790 Is Associated With Aspirin-Exacerbated Respiratory Disease: Replicative Study Using a Candidate Gene Strategy

“…An analysis of surrounding regions shows that this synonymous polymorphism (C) is within the rich zone of CpG islands (Li and Dahiya, 2002 ). This type of DNA region is strong, resistant to denaturation and is difficult to hybridize primers in conventional qPCR; therefore, it may not be the ideal technique for validating this finding (Flores-Juárez et al, 2016 ).…”