Overview

Oligo is a program for designing and analyzing synthetic oligonucleotide primers and genes. Based on nearest-neighbor thermodynamics, Oligo's search routines find optimal primers and primer sets for performing the polymerase chain reaction (PCR), including degenerate primers and primers suitable for multiplex PCR, inverse PCR, and site-directed mutagenesis; for designing hybridization probes; and for sequencing. To run a search, you can define a host of parameters and hybridization stringencies, or choose from among sets of predefined parameters. For each primer or primer pair, Oligo's various analysis windows show a multitude of useful data (such as length, composition, melting temperature, priming efficiency, potentials for duplex and hairpin formation, and internal stability) in graphical or tabular form.

You can also scan through a sequence manually using the Oligo Melting Temperature window. After completing a search, you can display all the potential primers in a list and sort them according to their position in the DNA sequence, product length (for PCR primer pairs), optimal annealing temperature, and CG content. Clicking on a primer (or primer pair) in the list automatically updates the data for that primer in all analysis windows. You can choose particular primers from the search and assign them to a primer database or to a "memory table" for future reference, and even enter the oligo sequence into an order form that you can fax or email to your favorite vendor of synthetic oligos.

Available platforms	Macintosh; Microsoft Windows 95, 98, and NT
System requirements	Macintosh Required: Any 68030-based computer with 4 Mb RAM or more, running System 7 Recommended: Power Macintosh with 8 Mb RAM or more, running System 8; large-screen color monitor Windows Required: 486 or higher with approximately 4 Mb RAM, running Windows 95 or higher Recommended: Above plus large-screen color monitor
Test platforms	Power Computing PowerTower Pro (604e), 225 MHz with 96 Mb RAM and 2 Gb hard drive; 20-inch color monitor
	Power Macintosh G3, 300 MHz with 128 Mb RAM and 9-Gb hard drive; 17-inch color monitor
Price	$1,200 $960 nonprofit (see vendor information)

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

About a week going through the tutorials and exploring with my own sequence files.

Product Quality

Customizability

Primer searches can be customized to almost any parameter. Plots of melting temperature, internal stability, and degeneracy can be displayed as histograms or line graphs, and can be resized. Each user can save his or her work so that the same set of windows appears in the next session.

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

Scripting is not available. Oligo does permit you to add external files, such as restriction enzyme lists and 6- and 7-mer frequency tables, codon bias tables, and repetitive sequences from various organisms, but importing them requires manual editing of text files.

Ability to Import and Export in Different File Formats

Oligo can open a wide variety of DNA, RNA, and protein sequence file formats, including GenBank, EMBL, and plain text. Sequences and data generated in Oligo can be exported as text files to any word-processing or text-editing program (designated in the Preferences menu).

Useful or Unusual Features

Although Oligo has many ancillary features, its forte is picking the oligos that are most likely to work in your PCR experiments. There are many variations on the basic PCR theme, and Oligo has tools to handle most of them. I wish I'd owned Oligo about a year ago when I was using multiplex PCR to do multifactor genetic mapping of the nematode gene I'm studying. In multiplex PCR, you use several primer pairs in a single reaction. Even with standard two-primer PCR, one oligo can interfere with the priming efficiency of the other oligo, and with multiplex PCR, the probability of primers not getting along with each other is heightened; picking a set of multiplex primers by eye is essentially hit or miss. Oligo's multiplexing feature can search through all the primers you have selected and build a set in which every primer is compatible with every other primer.

For investigators who want to clone a gene by heterology, starting with a gene derived from another organism, Oligo can design consensus oligos using multiple sequence files from different organisms or gene families. This is a new feature of version 6, added in response to user demand. Alternatively, you may want to do the converse experiment - target a specific member of a multi-gene family. Oligo can search through the sequence files for all family members and suggest optimal primers to amplify the member you want and ignore the rest. The "Priming Efficiency" algorithm will even give you a quantitative measure of the primer's specificity and efficiency.

To eliminate the problem of false priming, Oligo includes a set of files of organism-specific repetitive sequences, as well as frequencies of 6- and 7-mers in various organisms. When performing a search, you can elect to screen potential primers against these sequences.

Some other techniques for which Oligo is useful include site-directed mutagenesis, the ligase chain reaction (LCR), and various methods of reverse translating protein sequences. Oligo can find all potential restriction sites from a protein sequence. It displays important data, such as strongest hairpin structures and codon preference, as you edit the sequence - features that are very useful for designing synthetic genes. Underlying all of Oligo's primer-searching tools is a sophisticated data management system for keeping track of, and even ordering, all those handy primers.

Limitations

Oligo is not a program that you can sit down and learn to use simply by browsing through menu options, though the software's power makes climbing its learning curve well worth the effort. Nor is it foolproof: as pointed out in the user manual, there are some situations in which you can inadvertently corrupt a sequence you are working on. For example, if you do not complete one set of steps in the correct order, you can inadvertently "double-reverse translate" your sequence. The program should include safeguards, or at least offer warning dialog boxes, to prevent this sort of user error. Fortunately, Undo is available to fix the mistake.

Another limitation is that although Oligo has a basic restriction mapping feature, it is much less sophisticated than similar functions in other software packages. You can select restriction sites from a list, but you cannot filter sites according to any other criteria, such as the number of cuts or type of overhang. Also, you cannot pick an individual restriction enzyme out of a list for mapping; to do this, you must type the restriction site sequence into the String Search window. Oligo allows you to edit a selected primer, for instance, to add a restriction site to the 5' end. However, the program doesn't keep track of the altered position of the new primer on the original sequence; you must count and type in the new start position yourself.

Comparisons with Previous Versions and Similar Software

There are a number of packages, both commercial and free, for analyzing PCR primers. Many programs will analyze defined primers, but do not select optimal primers based on a DNA sequence. There are some Web-based primer selection services, such as Primer3 and Primers!, that have many, but not all, of the same features as Oligo, although with a more spartan user interface. Oligo has perhaps the best combination of analytical features, displays, speed, user interface, and database capabilities of any of them. Of course, this broad range of functions comes at a price.

Technical Support and Documentation

The user manual is the primary means for learning Oligo. Aside from a point-by-point description of all the menus and windows, the user manual contains a 30-page tutorial. While the tutorial is essential for learning the program, I found it inadequate for mastering all of Oligo's features. Many of the tutorial examples did not use real sequence files, but were just generic instructions. Often the program did not behave as the tutorial said it should. For all the shortcomings of Oligo's documentation, one very welcome aspect of the user manual is the appendix, which contains a thorough, clear discussion (including primary references) of the theoretical considerations the program uses to pick and analyze primers.

Online help in the Macintosh version of Oligo is in the form of help balloons, but these are only for menu items, not for the graphical program elements. Oligo offers free technical support for registered users by email and phone (toll free). Technical support is prompt, thorough, and courteous, and you will likely talk to the author of the software himself. Oligo's Web site provides downloadable software updates and demos, and has a guided tour of the program with annotated screen images.