Microarray Probe Design

From a probe design perspective, there are two types of oligonucleotide microarrays utilizing short (up to 25 nt) or long (up to 60 nt) probes. Long probes are major successors of cDNA-s and PCR amplicons utilized for expression profiling and whole genome comparisons, whereas short probes are good for single nucleotide polymorphism analysis genotyping and identification of microbial pathogens.

Selection of proper probes is one of the key steps for obtaining consistent and reliable data from microarray hybridization experiments. The main goals in probe design are common for all platforms and can be summarized as high specificity (complementary to the unique region of the sequence of interest) and uniform hybridization properties for all of the probes in the array. Attainment of these design goals provides accurate detection and quantification of the target over a large dynamic scale.

To satisfy these design goals, the typical range of GC content should be between 40% and 60% to avoid the presence of long GC clusters that could cause unspecific binding. Repetitive motifs or regions consisting of the same consecutive bases may result in slippage along the target resulting in unspecific hybridization. Self-annealing sites allowing hairpin formation have the potential to reduce the number of available probes for hybridization. Common practice is to eliminate secondary structure concerns by using denaturing solutions during the spotting step (50% DMSO) and/or high stringency conditions for the hybridization step (20-50% formamide or 55-65°C).

Whereas these rules of probe design are reasonable and straightforward, still, there is no simple model for a priori prediction of hybridization intensities that produces consistent estimates of hybridization behavior. Several parameters such as probe GC content, length, base order, and even kind of terminal nucleotides, as well as size, type (DNA or RNA), and secondary structure of the target could influence both the thermodynamics and kinetics of probe-target duplex formation. Additionally, hybridization conditions (temperature, time, ionic strength, concentration of the probe and the target, the presence of denaturing agents or detergents) and washing conditions (temperature and ionic strength) contribute to final signal intensity on microarrays.

One of the most promising approaches for probe design is based on the nearest-neighbor model described above. This model postulates that the stability of DNA-DNA duplexes mainly depends on the composition and orientation of neighboring base pairs. The duplex stability is correlated with a value of target-probe hybridization free energy (AG), which can be calculated from entropy and enthalpy of each possible matched nearest-neighbor pair. Despite the fact that values used in the nearest-neighbor model were obtained for hybridization in solution, calculated free energies of duplexes demonstrate a clear correlation with the hybridization intensities on microarrays. Probes with similar AG values of duplex formation are found to bind their corresponding targets with equal intensity.

Methods for probe synthesis and immobilization on a chip surface must be taken into account when discussing quantitative reliability of DNA-microarray data. Recent advances in technology allow synthesis of high-density arrays of probes (up to 106 probes/cm2) directly on a chip surface using photolithography. However, the chemistry of the in situ synthesis has a finite yield such that in a 25-step reaction with 99% yield at each step, the resulting yield of probes with the correct sequence is only 78% (0.9925 = 0.78). Alternative array manufacturing approaches such as ink-jet printing and robotic spotting each produce microarrays with different hybridization properties.

Microarrays of probes attached to a chip surface by either their 3'- or 5'-ends appear to have nearly identical sensitivity, specificity, dynamic range, and reproducibil-ity. At the same time, probes synthesized on a chip surface in 3'! 5' direction or attached to a glass slide through 3'-ends cannot be utilized in many common types of enzymatic modifications such as primer extension, liga-tion, and flap cleavage.

Selection of proper length of probes is dictated by the experiment objective. In general, long probes (50-60 nt) produce more intense hybridization signal than shorter ones but possess lower specificity. Intensity of the hybridization signal rises rapidly for probe lengths in the range of 17-50 nt and then reaches a plateau. The presence of a linker (typically 6-9 carbon atoms) between the chip surface and probe sequence facilitates hybridization for short probes that increases hybridization signal and results to higher sensitivity.

Single nucleotide polymorphisms (SNP) have increasing importance in both clinical and diagnostic roles. When designing a probe, some additional design rules should be applied for the best resolution of perfect match (PM) and single base pair mismatch (SBM) situations. In general, mismatch stability (Tm or AG of the duplex) is dependent on the length of probe, nature of the mismatch, mismatch position along the probe, and nearest neighbors of the mismatch. For shorter probes, the destabilizing effect of a mismatch is more dramatic. A reasonable compromise between specificity and sensitivity of SBM analysis can be achieved by using shorter probes (18-20 nt). The type of a mismatch is even more important than its location along the probe.

For example, in case of a G:A mismatch, reliability of the assay can be increased by switching to the antisense strand. The resulting C:T mismatch has a higher destabilizing effect, and the decrease in fluorescence intensity is more readily detected. Mismatch location is also an important concern in designing probes for SNP detection. In general, mismatches located near the ends of a probe have smaller destabilizing effect on the duplex. As a rule, ultimate and penultimate mismatches cannot be reliably detected even after optimization of hybridization and washing conditions.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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