Increased Single‐Nucleotide Discrimination in Allele‐Specific Polymerase Chain Reactions through Primer Probes Bearing Nucleobase and 2′‐Deoxyribose Modifications

Abstract

The diagnosis of genetic dissimilarities between individuals is becoming increasingly important due to the discovery that these variations are related to complex phenotypes like the predisposition to certain diseases or compatibility with drugs. The most common among these sequence variations are single-nucleotide polymorphisms (SNPs). The availability of reliable and efficient methods for the interrogation of the respective genotypes is the basis for any progress along these lines. Many methods for the detection of nucleotide variations in genes exist, in which amplification of the target gene is required before analysis can take place. The allele-specific polymerase chain reaction (asPCR) combines target amplification and analysis in a single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes. The most important parameter in asPCR is the discrimination of these matched or mismatched pairs. In recent publications we have shown that the reliability of SNP detection through asPCR is increased by employing chemically modified primer probes. In particular, primer probes that bear a polar 4'-C-methoxymethylene residue at the 3' end have superior properties in discriminating single-nucleotide variations by PCR. Here we describe the synthesis of several primer probes that bear nucleobase modifications in addition to the 4'-C-methoxymethylenated 2'-deoxyriboses. We studied the effects of these alterations on single-nucleotide discrimination in allele-specific PCR promoted by a DNA polymerase and completed these results with single-nucleotide-incorporation kinetic studies. Moreover, we investigated thermal denaturing of the primer-probe-template complexes and recorded circular dichroism (CD) spectra for inspecting the thermodynamic and photophysical duplex behaviour of the introduced modifications. In short, we found that primer probes bearing a 4'-C-methoxymethylene residue at the 2'-deoxyribose moiety in combination with a thiolated thymidine moiety have synergistic effects and display significantly increased discrimination properties in asPCR.

Chiral Selectivity in the Binding of [4]Helicene Derivatives to Double‐Stranded DNA

“…The data presented in Figure 3 were analysed using the McGhee and von Hippel model [Eq. (2)]. [25] The best fits of Equation (2) to the dependence of r/A C H T U N G T R E N N U N G [HelR] on r are shown in Figure S3 and the so-obtained binding constants, K, together with the number of base pairs perturbed by a bound dye, n, are listed in Table 2.…”

“…One way to overcome this complication is to study the interaction of DNA with small chiral organic molecules that are easier to access. [1] Moreover, molecules that are able to bind strongly and specifically to DNA can be potentially used in therapeutical applications, [2] medical diagnostics, [3] and genetic screenings. [4] Over the last decades, the interaction between numerous drugs and different DNA sequences and conformations has been intensively studied.…”

“…DNA binding constants (K) and number of base pairs involved in the binding (n) for the HelR enantiomers determined by using Equation(2).…”

Fabrication of biosensors using vinyl polymer-grafted carbon nanotubes

“…Currently, direct detection of nucleic acids is an area of tremendous interest, as it plays a major role in forensics (Jelly et al, 2008), pharmaceutical applications (Kranaster & Marx, 2007), medical diagnosis (Marras et al, 2006), food and agricultural analysis (Timko et al, 2008), and environmental control (X. C. Shen et al, 2008). Electrochemical transducers offer many potential advantages that include sensitivity, accurate specificity, simplicity and lowcost, in converting nucleic acid hybridization events into useful analytical signals (Millan et al, 1994;Hashimoto et al, 1994;S.…”

Selectivity of Enzymatic Conversion of Oligonucleotide Probes during Nucleotide Polymorphism Analysis of DN

“…The 3’–terminus of the oligonucleotide primer was modified with a thymidine analog bearing a 4’– С –methoxymethylene residue in its deoxyribose, as well as a thiol group in the С 2 or С 4 position of the thymidine base [109]. Reactions with the Vent DNA–polymerase and primers with a single modification exhibited increased discrimination only with the use of a carbohydrate–substituted oligonucleotide ( T OMe , Δ С t = 9).…”