Overview

Primer Premier 5 integrates multiple-sequence alignment with primer design to facilitate the design of primers for amplification. A proprietary algorithm is used to calculate a minority consensus that uses degenerate bases to represent all possible bases in a particular sequence position. Based upon this consensus, primers are designed in highly conserved regions of the sequences. Any sequence in the alignment, or any unsequenced species and pathogens, may be amplified using the resultant primers. Allele-specific primers of the minority sequence of a gene family can be designed graphically.

Primer Premier provides comprehensive primer-design features that allow primers to be designed automatically or manually, and multiplex/nested primer design ensures no cross homologies. Other features are a primer database for storage, a synthesis order form to process ordering, and the easy editing of primers for site-directed mutagenesis and the creation of enzyme sites. Also, sequences can be analyzed for restriction-enzyme and motif sites.

How Long Did It Take to Learn to Use It Productively?

One to two hours. After taking the 15-minute tour and exploring the menu items, I was able to start designing primers immediately. For more advanced functions, up to one day to experiment with different menus is required.

Product Quality

Customizability

The program is customizable in that many options are available for choosing what data is stored in the database and printed. Even the address of your standard synthesis facility for ordering primers is stored after you key it in once. The last enzyme set tested is stored to reflect enzymes available prior to new testing.

Ability to Program in Scripts, Add Extension Modules, etc.

Not applicable.

Ability to Import and Export in Different File Formats

Any sequence in Genbank format can be input. In addition, sequences from any other sources can be loaded as long as they are available in text format. Pasting sequences or fragments using the clipboard is available as well. Many reports are available for output. Oligo ordering forms can be quickly generated using the primer order facility provided.

Useful or Unusual Features

Cross-Species Primers

Primer 5 is the first program to integrate fully multiple-sequence alignment (ClustalW) with primer design to facilitate the design of primers that can be used for cross-species, pathogenic, or allele-specific research. The alignment can be done on the Web and then imported into the program, or it can be done directly in the program. The Macintosh version, however, does not support local alignment. The primers are shown on the alignment, allowing easy visualizing of primer/template mismatches. Primers for detection of mutagenic bases can be easily designed by clicking on any base to evaluate the primer whose 3' end starts from that base.

Primer Search

The primer search can be done in automatic mode, allowing for rapid computer selection of optimal primers, or in manual mode, allowing fine manual control of the primer's physical characteristics.

In the automatic search mode, the program rapidly selects optimal primers using preset physical and chemical parameters including Tm, GC percent, degeneracy, presence of secondary structures, primer length, and thermodynamic profile. The program automatically starts with a high stringency and progressively lowers the stringency until primers are found. It then displays in Primer Analysis the primers in ranked order based on their primer rating and including all of the data listed above. Primers can be searched for physical characteristics suitable for hybridization, sequencing, or PCR.

For users who require a fine degree of control over primer selection for specialized PCR processes, a manual search is available. In the manual search, the user can specify exact physical characteristics for the primer. All primers meeting these criteria within the specified region of the target region will be displayed. Each criterion used in the manual selection can be individually controlled.

Sequence Editor/Translator

Primer Premier contains a fully featured sequence editor capable of handling sequences up to 50 kb long. Sequences can be pasted into the editor while complementing, reversing, or reverse/complementing, making it easy to get the desired orientation. Sequences can be proofread using multimedia voice readback, facilitating the entry of sequences from manual sequencing runs.

Forward and back translation between DNA sequences and protein sequences is provided. Twenty mitochondrial codon tables are available for use in translation, as well as editing facilities so that the user can enter additional codon tables. Back translation of protein sequences enables degenerate-primer selection.

Degenerate-Primer Design

Because of redundancy in the genetic code, oligonucleotide design must account for ambiguous DNA bases. Typically, in DNA sequences (obtained from standard or personalized codon tables) determined from a reverse-translated peptide, as many as 50 percent of the bases may be ambiguous. To lessen this ambiguity, primers must be designed from regions of low degeneracy. Primer Premier is adequately equipped to scan automatically target-DNA sequences and design primers for regions of low degeneracy that are free of secondary structures such as hairpins, dimers, and false priming sites. This method is time efficient compared to manual searching of the sequence using a degeneracy graph.

Restriction-Endonuclease and Motif Analysis

A database of 900 common enzymes and 150 motifs is contained within Primer Premier 5. This module contains a wide selection of enzyme-sensitivity and motif-recognition algorithms. The enzyme set for analysis can be selected manually or with the powerful filtering capability. Enzymes can be filtered by several parameters, including the number of recognition sites and the overhang sequence. The results can be viewed as a map, table, or sequence configuration.

Motif analysis of a selected sequence is provided by a concise account of the number of recognizable motifs, their positions, and a short description of their distinctive attributes. Similar to the enzyme analysis, new motifs may be added with tools from the GeneTank window.

Nested Primers

To design primers for single-tube multiplex protocol, a set of primers screened for Tm and secondary structures for the entire sequence can be displayed graphically. Using the graphical selection mechanism, a set of primers free of cross-dimers can be selected easily.

Primer Database

The primer database allows multiple users to have their own primer database containing primer sequences. This makes it easy to track the oligos contained in a lab while being able to separate them by category. Oligos in the database can be exported into the program for analysis on any target sequence. This makes it simple to test whether primers designed for one gene will amplify a related gene.

Limitations

The major limitation is lack of support for the ligase chain reaction. The program provides no ability to design primers for this important protocol. The Macintosh version does not support sequence alignment locally, and the multimedia tour cannot run on Macintosh computers.

Comparisons with Similar Software

Compared to programs such as Oligo or PrimerSelect, Primer Premier is cheaper and easier to use. The cross-species primer-design facility in version 5 is a significant addition to the previous version of Primer Premier.

Technical Support and Documentation

The ease of using Primer Premier is due in part to the multimedia tour that one can download from the Web site. The tour is free. It provides a good introduction to the main features, and is an effective way to learn to use the program.

The online help was quite useful and timely. The manual is well written, covering every function in a concise manner. Discussions of the criteria that go into the design and analysis of an effective primer form a welcome section.

Technical support is freely available through email and phone. Support staff are effective, helpful, and knowledgeable.

Target Users

Any researcher or laboratory that designs oligos on a regular basis will find this software useful. This software is a good tool to design cross- species, pathogenic, or allele-specific primers, to run single-tube nested PCR reactions, or to amplify a gene based solely on an amino-acid sequence. The lower price and rich functionality make this product possibly the best value available.