Tuning single nucleotide discrimination in polymerase chain reactions (PCRs): synthesis of primer probes bearing polar 4'-C-modifications and their application in allele-specific PCR

There are many methods available for the detection of nucleotide variations in genetic material. Most of these methods are applied after amplification of the target genome sequence by the polymerase chain reaction (PCR). Many efforts are currently underway to develop techniques that can detect single nucleotide variations in genes either by means of, or without the need for, PCR. Allele-specific PCR (asPCR), which reports nucleotide variations based on either the presence or absence of a PCR-amplified DNA product, has the potential to combine target amplification and analysis in one single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes by using allele-specific primer probes. PCR amplification by a DNA polymerase from matched 3'-primer termini proceeds, whereas a mismatch should obviate amplification. Given the recent advancements in real-time PCR, this technique should, in principle, allow single nucleotide variations to be detected online. However, this method is hampered by low selectivity, which necessitates tedious and costly manipulations. Recently, we reported that the selectivity of asPCR can be significantly increased through the employment of chemically modified primer probes. Here we report further significant advances in this area. We describe the synthesis of various primer probes that bear polar 4'-C-modified nucleotide residues at their 3' termini, and their evaluation in real-time asPCR. We found that primer probes bearing a 4'-C-methoxymethylene modification have superior properties in the discrimination of single nucleotide variations by PCR.     

Structurally and chemically heterogeneous nanofibrous nonwovens via electrospinning

Homogeneous nonwovens composed of polymer nanofibers of a given diameter are characterized by structural parameters such as the average pore sizes and internal surfaces as well as by transport properties, which are strongly correlated to the fiber diameter at a given porosity. Such nonwovens are used among others for filter applications, protective clothing or as scaffolds for tissue engineering. A frequent requirement is that, to be able to prepare nonwovens optimised for the specific application, one has to find ways to disrupt this strong correlation allowing independent modification of pore diameter, transport properties and internal surface or to induce local chemical and structural heterogeneities within the nonwoven. The route explored in this paper is based on the electrospinning of heterogeneous nonwovens composed of nanofibers with two different average diameters (by a ratio of up to 10 and more) on the one hand and/or different chemical nature on the other hand. Spinning parameters have been optimised to achieve this goal. In addition, nonwovens composed of fibers with circular cross-section and with ribbon-like cross-section have been prepared.     

Mass spectrometric diagnosis of the surface nitriding mechanism in a d.c. glow discharge

Reactive constituents have been investigated in a molecular beam generated in the cathode surface glow area and surface boundary layer. Mixtures of nitrogen and hydrogen form NH_x(x=0–4) compounds, which are of relevance in heterogeneous, plasma vs. metal nitriding reactions. Ammonia decomposition leads to NH_x(x=2–4). Strong cataphoretic enrichment of hydrogen has been observed in the cathode glow area. Heterogeneous reactions of NH_x with iron lead to the formation of iron nitrides via intermediates such as FeNH_2–3. In a pulsed d.c. glow discharge, increased sputtering and decreased hydrogen enrichment have been observed.     

An optimal two-step quadratic spline collocation method for the Dirichlet biharmonic problem

Numerical Algorithms - A two-step quadratic spline collocation method is formulated for the solution of the Dirichlet biharmonic problem on the unit square rewritten as a coupled system of two...     

The job-shop problem and immediate selection

The job-shop problem is one of the most difficult NP-hard scheduling problems. A 10×10-problem published in 1963 has been solved only recently by Carlier and Pinson using a branch and bound method. Other branch and bound algorithms have been developed recently. The efficiency of all these branch and bound methods relies on the concept of immediate selection which allows to introduce order relations on the setI of all operations to be processed on the same machine before branching. We present new algorithms for immediate selection. Among them are

1. anO(max {n logn,f})-algorithm for fixing all disjunctions induced by cliques;
2. anO(n ²)-algorithm based on concepts which are different from those used by Carlier and Pinson.

Here,n is the number of operations inI andf is the number of induced order relations.     

The Lewis basicity of the [Pt3(μ-CO)3(PR3)3] clusters and the X-ray crystal structure of [{ZnI2}{Pt3(μ-CO)3(PPh(i-Pr)2)3}]

The cluster compounds [Pt₃(μ-CO)₃(PR₃)₃] act as Lewis bases towards the metal halides of Group XI, MX, Group XII, MX₂, and Group XIII, MX₃, to form cluster compounds of the composition [{MX_n}{Pt₃(μ-CO)₃(PR₃)₃}]. The X-ray crystal structure, NMR and IR data are given for the compound [{ZnI}{Pt₃(μ-CO)₃(PPh(i-Pr)₂)₃}].     

The Structures of Some Products from the Photodegradation of the Pluramycin Antibiotics Hedamycin and Kidamycin

The photolability of the antitumor antibiotic hedamycin ( 1 ) was investigated by irradiation in different solvents in the presence or in the absence of oxygen. The products formed were separated chromatographically and their structures determined by NMR spectroscopy. Photolysis of 1 in the presence of oxygen gave only one isolable product, photohedamycin A ( 3 ), where ring E of hedamycin had been transformed into an enol ether. The reaction in the absence of oxygen yielded the photohedamycins B, C, and D ( 5, 6 , and 7 , respectively). In these compounds, one of the epoxides of hedamycin had been opened reductively, and in photohedamycin D ( 7 ) the substituent at C(8) - originally ring E of hedamycin - was now acyclic. In addition to these compounds, the photolyses yielded a large number of unstable minor products, which could not be isolated.     

Neutron activation analysis of heavy rare earths

A method of nondestructive activation analysis of heavy rare earth elements (Sm−Lu) is described. Intensities of γ-ray peaks are compared to that of an internal reference after irradiations by both thermal and fission spectrum neutrons. Results are obtained from computer comparisons of peak areas. Work was performed at the Ames Laboratory of the U.S. Atomic Energy Commission. Contribution No. 2895.     

NMR spectra of a microcrystalline protein at 30 kHz MAS

Proteins are not always available in amounts desirable for solid-state magic-angle spinning (MAS) nuclear-magnetic resonance (NMR) spectroscopy. To maximize the signal-to-noise ratio achievable with small samples, the filling factor must be optimized by using small-diameter MAS rotors. These rotors have the added benefit of allowing higher radio frequency field amplitudes during polarization transfer steps and during decoupling periods as well as allowing higher spinning frequencies. We demonstrate the advantages of relatively fast MAS (30 kHz using a 2.5 mm rotor) compared to MAS at 12 kHz for the 10.4 kDa model protein Crh with 93 residues and show that the signal-to-noise ratio in two-dimensional correlation spectra can be significantly improved by taking advantage of optimized pulse sequences available with rapid MAS.     

Synthesis of Naturally Occurring Pyrazine and Imidazole Alkaloids from Botryllus LeachiRID=”a”ID=”a”?Dedicated to Prof. G. M?rkl on the occasion of his 75th birthday

The synthesis of the naturally occurring and biologically active alkaloids 1 and 2, first isolated from the red ascidian Botryllus leachi by Duran et al. [1], is described and the structure proposed for Botryllazine B (1) is confirmed. The analytical data for 2-(p-hydroxybenzoyl)-4-(p-hydroxyphenyl)imidazole (2) are discussed and compared with the literature. With special emphasis of ¹H NMR data the tautomerism of aroylimidazolemethanones is described.