Algorithm For Oligonucleotide Design

Software for oligonucleotide design is commonly available1-5,6-1 and generally has input, sequence analysis, and output modules. An outline of such an algorithm for oligonucleotide design is shown in Fig. 1.

In the input module, the user specifies the target sequence and various parameters for the desired oligonu-cleotides including melting temperature range, salt concentration, and the length range. Other input parameters (not shown) may include weighting factors for the various design constraints under consideration. The analysis module first carries out input manipulation, reading the target sequences and performing multiple alignment of sequences if the genes analyzed are part of a gene family or include conserved domain. The alignment allows design of PCR primers for amplification of multiple conserved sequences and the development of oligonucleotides that can either detect all conserved sequences or distinguish among conserved sequences.

A first element of the sequence analysis is the Tm calculation for each candidate oligo using the nearest-neighbor model. Each sequence is then scored for potential secondary structures, such as hairpins and self-complementary regions, to limit self-annealing. Alignment between the candidate oligo and the target (local alignment) is performed to look for and avoid other hybridization sites.

The sequence analysis module may perform additional analysis steps for oligonucleotides to be used as PCR primers, including pair selection of forward and reverse primers using all of the permutations of the candidates. Amplicon length is calculated for each primer pair along with the melting temperature of each primer. Additional rules for primer selection such as 3'-end requirements can be applied at this point to find preferred primer pairs. For oligonucleotides to be used as probes, SNP analysis and selection of overlapping primers (tiling) may be performed.

Finally, the output module produces a list of oligonu-cleotides with their sequences, location on the target sequence, and melting temperature, and may include a map of the target sequence indicating the position of the oligonucleotides.

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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