Multiplex qPCR assay for identification and differentiation of Amblyomma americanum, Amblyomma cajennense, and Amblyomma maculatum (Ixodida: Ixodidae) tick species in the eastern United States.

Many ticks of the genus Amblyomma are vectors of human pathogens, and the correct species identification is medically and epidemiologically important. Morphological identification is time-consuming and requires a high level of expertise. Identification of engorged, immature, or damaged ticks and the differentiation of closely related species remain problematic. Here, we report the development of a real-time TaqMan assay for the genomic identification and differentiation of Amblyomma americanum (L.), Amblyomma cajennense (F.), and Amblyomma maculatum (Koch), which are human-biting species found in the eastern United States. New species-specific sets of oligonucleotides for the multiplex reaction that detect and differentiate the ITS2 genomic regions of three target species were designed using Visual OMP; the previously published A. americanum oligonucleotide set was also incorporated into our assay. Specificity and sensitivity tests for two multiplex master mixes using different A. americanum sets were performed using individual and pooled samples of adult, nymphal, and larval ticks, and optimization procedures were applied. The multiplex assay successfully differentiates between genomes of three target species and does not cross-react with DNAs of ticks from other genera. Rare cases of nonspecific amplification occurred with DNAs of A. imitator and Amblyomma triste Koch misidentified as A. americanum and A. maculatum, respectively. However, this cross-reaction does not diminish the usefulness of the developed assay east of the 95th meridian, where neither A. imitator nor A. triste are found. Two master mixes incorporating the previously published or newly developed A. americanum sets are being recommended for identification of individual ticks or pooled samples, respectively.

Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes.

The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°(37), ΔH° and melting temperature (T(m)) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG > rU·dT > rA·dA > rC·dC. The stability trend for the base pairs 5' of the single internal mismatch is rG·dC > rC·dG > rA·dT > rU·dA. The stability trend for the base pairs 3' of the single internal mismatch is rC·dG > rG·dC >> rA·dT > rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST.

Hachimoji DNA and RNA: A genetic system with eight building blocks.

We report DNA- and RNA-like systems built from eight nucleotide "letters" (hence the name "hachimoji") that form four orthogonal pairs. These synthetic systems meet the structural requirements needed to support Darwinian evolution, including a polyelectrolyte backbone, predictable thermodynamic stability, and stereoregular building blocks that fit a Schrödinger aperiodic crystal. Measured thermodynamic parameters predict the stability of hachimoji duplexes, allowing hachimoji DNA to increase the information density of natural terran DNA. Three crystal structures show that the synthetic building blocks do not perturb the aperiodic crystal seen in the DNA double helix. Hachimoji DNA was then transcribed to give hachimoji RNA in the form of a functioning fluorescent hachimoji aptamer. These results expand the scope of molecular structures that might support life, including life throughout the cosmos.

Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes.

Nearest-neighbor thermodynamic parameters of the 'universal pairing base' deoxyinosine were determined for the pairs I.C, I.A, I.T, I.G and I.I adjacent to G.C and A.T pairs. Ultraviolet absorbance melting curves were measured and non-linear regression performed on 84 oligonucleotide duplexes with 9 or 12 bp lengths. These data were combined with data for 13 inosine containing duplexes from the literature. Multiple linear regression was used to solve for the 32 nearest-neighbor unknowns. The parameters predict the T(m) for all sequences within 1.2 degrees C on average. The general trend in decreasing stability is I.C > I.A > I.T approximately I. G > I.I. The stability trend for the base pair 5' of the I.X pair is G.C > C.G > A.T > T.A. The stability trend for the base pair 3' of I.X is the same. These trends indicate a complex interplay between H-bonding, nearest-neighbor stacking, and mismatch geometry. A survey of 14 tandem inosine pairs and 8 tandem self-complementary inosine pairs is also provided. These results may be used in the design of degenerate PCR primers and for degenerate microarray probes.

New prediction model for probe specificity in an allele-specific extension reaction for haplotype-specific extraction (HSE) of Y chromosome mixtures.

Allele-specific extension reactions (ASERs) use 3' terminus-specific primers for the selective extension of completely annealed matches by polymerase. The ability of the polymerase to extend non-specific 3' terminal mismatches leads to a failure of the reaction, a process that is only partly understood and predictable, and often requires time-consuming assay design. In our studies we investigated haplotype-specific extraction (HSE) for the separation of male DNA mixtures. HSE is an ASER and provides the ability to distinguish between diploid chromosomes from one or more individuals. Here, we show that the success of HSE and allele-specific extension depend strongly on the concentration difference between complete match and 3' terminal mismatch. Using the oligonucleotide-modeling platform Visual Omp, we demonstrated the dependency of the discrimination power of the polymerase on match- and mismatch-target hybridization between different probe lengths. Therefore, the probe specificity in HSE could be predicted by performing a relative comparison of different probe designs with their simulated differences between the duplex concentration of target-probe match and mismatches. We tested this new model for probe design in more than 300 HSE reactions with 137 different probes and obtained an accordance of 88%.