Use of Base-Modified Duplex-Stabilizing Deoxynucleoside 5′-Triphosphates To Enhance the Hybridization Properties of Primers and Probes in Detection Polymerase Chain Reaction

Abstract

Several base-modified duplex-stabilizing deoxyribonucleoside 5′-triphosphates (dNTPs) have been evaluated as agents for enhancing the hybridization properties of primers and probes in real-time polymerase chain reaction (PCR). It was shown that pyrimidines substituted at the 5-position with bromine or iodine atoms and methyl or propynyl groups are incorporated into PCR amplicons by Taq DNA polymerase as efficiently as natural dNTPs. The dNTP of 2-aminoadenosine was incorporated somewhat less efficiently than dATP but still supported PCR. Incorporation of these modified nucleotides into the amplified DNA represents a simple and inexpensive way to stabilize duplexes of primers and probes and is particularly effective in improving the amplification and detection of A/T-rich sequences. This technology permits the use of higher PCR annealing temperatures or alternatively a reduction in the length of the oligonucleotide components. Examples of successful application in TaqMan and Scorpion real-time detection assays are provided. Limits of the approach are identified and discussed. For example, application of the 5-bromo and 5-iodo derivatives may be limited to relatively G/C-rich DNA targets and, in particular, to those lacking long runs of adenylate and/or thymidylate. Simultaneous use of base-modified analogues of dATP and dTTP should be avoided in PCR due to "overstabilization" of the amplicon.Structurally modified DNA and RNA have applications in bioengineering, nanotechnology, molecular biology, and medicine. Oftentimes, however, chemical synthesis of modified polynucleotides is difficult and inefficient. Moreover, certain modifications cannot be chemically introduced due to instability. Enzymatic synthesis is an alternative way to prepare DNA or RNA polymers. Relatively minor modifications at the R-phosphate moiety of deoxyribonucleoside 5′-triphosphates (dNTPs) are tolerated well by DNA polymerases (1). Examples include thio (2) and borano phosphate (3, 4) derivatives. DNA polymerases also tolerate certain base modifications in dNTPs as long as normal Watson-Crick base pairing is preserved.

N 6-Methyladenine hinders RNA- and DNA-directed DNA synthesis: application in human rRNA methylation analysis of clinical specimens

“…S1 † ) were used. 18 As shown in Fig. S5 and S6, † a consistently lower incorporation of 5-halogenated dUTPs opposite DNA-m 6 A was characterized, compared with DNA-A.…”

Synthesis of Oligodeoxynucleotides Using Fully Protected Deoxynucleoside 3′-Phosphoramidite Building Blocks and Base Recognition of Oligodeoxynucleotides Incorporating N3-Cyano-Ethylthymine

“…To study the template specific incorporation of dNTPs using DNA polymerase, we examined the single insertion reaction of dNTPs toward the opposite T CE in templates using Taq polymerase [ 37 ], a well-known thermostable enzyme used in PCR reactions [ 38 , 39 ]. The template incorporating T or T CE at position X was annealed with a 5′-FAM-labeled 18-nt primer in the presence of DNA polymerase.…”

New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Förster resonance energy transfer probes in 5′-nuclease assays

“…Although the reported data indicate that this thermodynamic gap is sufficiently broad, a number of potential improvements to the Snake technology have already been sought. For example, preliminary results point to an advantage of using base-modified duplex-stabilizing dNTPs (33). Along with the anticipated benefit to the hybridization properties of PCR primers and probes, this approach would lead to a disproportional stabilization of the key sense amplicon in comparison to the problematic antisense one.…”