Design Of Degenerate Sequence Oligonucleotides

In some DNA microarray-based applications, ''universal'' primers, which enable amplification of several related amplicons simultaneously, can significantly reduce the number of reactions needed and thus save cost and time. The design of universal PCR primers relies on multiple sequence alignment (alignment of multiple sequences that inserts gaps into the individual sequences to align conserved sequences in the same column) which is used to identify sequences of highly conserved regions. Instead of developing individual primer sets for each of a group of paralogous genes or alleles of a single gene, universal primers can be designed to amplify several such genes simultaneously. Identification of the resulting amplicons by DNA microarrays allows discrimination of a range of genes using a single PCR reaction, as demonstrated for amplification and identification of 16 Staphylococcal enterotoxins simultaneously.[4]

As a rule, these universal primers are designed from conserved regions of related genes. In many cases, there are several positions within these conserved regions that exhibit variation. Universal primers can be designed, based on such regions, as a mixture of all the sequence permutations represented by the variation at each location. Such primers are termed degenerate and are described using the International Union of Pure and Applied Chemistry (IUPAC) base nomenclature.

For example, the sequence AHWRT represents a degenerate primer 5 bases long with degeneracy at positions 2-4 where H represents A, C, or T, W represents A or T, and R represents A or G.

During synthesis of this degenerate primer, a mixture of the indicated phosphoamidites is added into the reaction at the variable positions; for example, the second base in the example sequence can be A, C, or T. The synthesis results in a mixture of 5-nt-long primers instead of a single sequence. The degeneracy of the final primer mixture is the product of the degeneracy at each base position (degeneracy = 12 for the 5-nt example).

Degenerate primers have a range of melting temperature which can be calculated using the nearest-neighbor model. The lower limit of the range typically corresponds to the sequences formed by low melting bases (A-T) and can be recommended as the annealing temperature in initial experiments. A second concern regarding degenerate primers is concentration of the primers in a PCR reaction. The concentration of each individual sequence in a degenerate primer mixture is the total concentration divided by the degeneracy. Thus the primer concentration must be increased by a factor equal to the degeneracy to achieve the proper primer concentration for each individual primer in the mixture.

High values of degeneracy are impractical because of issues associated with primer concentration and the increased potential for primer dimers. Several rules can be applied to decrease the degeneracy of a primer designed for a particular set of conserved sequences. First, a few weakly destabilizing mismatches such as G:T can be ignored because they do not disrupt the duplex stability greatly. Second, universal nucleotide analogs can be used, such as inosine, which pairs stably with any of the four nucleotides. Because degenerate primers are actually mixtures of oligonucleotides, the experimental conditions (annealing temperature, buffer, magnesium chloride, primer concentration) should be optimized experimentally, similar to the procedure for multiplex PCR (PCR reaction which multiple DNA regions amplified simultaneously using multiple primers).

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.

Get My Free Ebook


Post a comment