PriFi: using a multiple alignment of related sequences to find primers for amplification of homologs

Abstract

Using a comparative approach, the web program PriFi (http://cgi-www.daimi.au.dk/cgi-chili/PriFi/main) designs pairs of primers useful for PCR amplification of genomic DNA in species where prior sequence information is not available. The program works with an alignment of DNA sequences from phylogenetically related species and outputs a list of possibly degenerate primer pairs fulfilling a number of criteria, such that the primers have a maximal probability of amplifying orthologous sequences in other phylogenetically related species. Operating on a genome-wide scale, PriFi automates the first steps of a procedure for developing general markers serving as common anchor loci across species. To accommodate users with special preferences, configuration settings and criteria can be customized.

Figure 1

For this sample alignment, two primer regions were identified (marked by +––+), using parameters minimum primer length = 18 and maximum number of ambiguities = 4. An asterisk symbolizes a perfect match, i.e. a column with at least 2 nt which are all identical. Gaps are ignored. The X'es in the first sequence indicate an intron of length <200 nt.

Figure 2

PriFi finds valid primer pairs using three filters (shown as three yellow triangles). The first filter operates on the alignment identifying highly conserved primer regions. The second filter identifies the individual primers within the primer regions, evaluates them according to certain criteria and discards those that are invalid. The third filter considers, evaluates and scores all possible pairings between valid primers, discarding invalid pairs.

Figure 3

With a minimum primer length of 18, one can place 10 different primers in a primer region of length 21: four of length 18, three of length 19, two of length 20 and one of length 21.

Figure 4

In the line overview at the top, the input sequences are the black lines while the suggested primer pairs and the corresponding PCR products each have their own, different color. The primers are the thickened ends of the lines. The sequence view below provides a close-up look at the alignment and the primers.