Test Platform

Macintosh Performa 5260 with 32 Mb RAM
Pentium 200 MHz with 64 MB RAM, with Windows 98, running Macintosh emulator Executor 2.0

Overview

Amplify is a Macintosh program that facilitates the selection, analysis, and simulation of Polymerase Chain Reaction (PCR) experiments. It allows one to analyze oligonucleotide primers for primer-dimer formation, and searches the target DNA sequence for homologous sections. Using this information, Amplify predicts the formation of PCR products, as well as the probability of the reaction that forms them.

Amplify uses sequence information for DNA templates and primers, and analyzes the binding sites to predict the part of the target that will be amplified. The program provides information about reaction products and primer binding sites; this information can help users design primers and plan PCR experiments. Amplify also checks the primer pairs for the formation of primer-dimers, which can cause PCR experiments to fail. The default settings for this analysis are based on empirical rules developed in the laboratory. Amplify allows experienced users to modify these settings when necessary. The software offers an excellent starting point for evaluating a PCR reaction, and serves as a great tool for students learning about PCR and molecular biology in general.

File Formats

Amplify supports DNA sequence information from any text file. One can copy and paste the template or target sequence, or simply open it directly from an existing file. When Amplify uses the target sequence (as by the Run PCR command, for example), the program converts the DNA sequence into a standard format. It removes characters other than A, T, C, and G, and arranges the sequence in 80-character lines. A header field, denoted by a user-selectable delimiter, may be appended to the sequence and is not reformatted. Many programs, such as DNA Strider, can export a sequence in a plain text format suitable for use in Amplify. Once one enters the target sequence, the Save As command creates a text file with the Amplify icon.

Amplify also stores the primer sequences in a plain text format. Each line of the list starts with the sequence of the primer (in the 5' to 3' direction), followed by a tab and the primer name. Users may append additional information to the same line after a second tab. Currently, Amplify limits the number of primers in the master list to 1,000. The name given to the primer list should end in ".pri", allowing the program to distinguish between a primer list and a sequence file. Primers used in PCR simulations are selected from this master list.

Using the Program

After selecting a target sequence and primers, users issue the Run PCR command to run a simulated PCR experiment. One simulation may use up to 20 primers; the Primers in Use window (figure 1) lists them. When Amplify simulates a PCR, it firsts formats the target sequence, then checks the selected primers for dimer formation. Next it searches the target sequence for sites complementary to the primers. Amplify renders this search in both directions, by taking the reverse complement of the selected primers and comparing them against the 5'-3' sequence. The program uses two criteria, primability and stability, to assess the likelihood of successful amplification. Primability is a term used to quantify how easily the DNA polymerase will be able to extend the sequence at the 3' end. In calculating primability, more weight is given to matches at the 3' end of the primer. Stability is a measure of how tightly the primer binds to the target sequence, regardless of where the matches occur in the primer sequence. In this regard, the program counts G-C pairs more heavily than A-T pairs, and runs of matches count more than single pairs.

Amplify expresses the primability and stability of a primer match as a percentage. One hundred percent primability is given to primers in which all nucleotides match. The amount of G-C pairs determines the stability score - only a primer with all G and C would have 100 percent stability. To achieve a successful amplification, the program's default cutoff values for primability and stability are 70 percent and 40 percent, respectively. Based on these results (figure 2), the program generates a graphical map of the likely amplification. This map shows all potential binding sites, possible fragments generated, and potential formation of dimers. The best matches and most likely PCR products are shown as dark, thick lines, while poor matches are light and thin. This display makes it easy for PCR experimenters to get a quick overview of probability for success (figure 3). Users may select all elements of the amplification map to show the corresponding detail in the Info window (figure 4). The resolution of the map may also be changed with the Zoom command in the PCR menu. This is especially useful when the program identifies a number of overlapping primer-binding sites.

One point of confusion may arise when Amplify uses a primer in the 3' direction. In this case, the information for that primer shows the reverse complement of the primer. It shows the target sequence only in the 5'-3' direction and the primer is aligned to this, making matches easier to see but also giving the "wrong" sequence. If one uses the Info window as the source for ordering an oligonucleotide primer, one must be sure to verify the correct sequence from the original list of primers.

Other Features

Amplify offers a number of other tools for working with primers. The Edit menu offers a quick way to convert between upper and lower case letters, while the Sequence menu can reverse the polarity of any sequence. Amplify also includes the commands to check single primers, or a list of multiple primers. If one selects a single primer, the Check this Primer feature will search for potential primer-dimers between this and all other primers in the list. When one selects Check All Primers, the program makes a table of all potential dimer-forming pairs from the loaded primer list (figure 5). Users can alter stringency for this analysis with the Change Dimer Parameters command.

Output Formats

Amplify offers two basic output formats. The first is simply a text information file, which users construct by clicking on different features of the amplification map. The second is the map itself - a graphical representation of the probable PCR products and primer binding sites. One can save the amplification map that Amplify generates as a PICT file, or copy it to the clipboard for pasting into another application. Alternatively, one can copy a portion of the map by holding down the command key, dragging the mouse so as to draw a rectangle around the region of interest, and then selecting Copy Pixels from the Edit menu. Although convenient, this method creates a pixel map which results in poor quality printed images.

Documentation

Amplify offers effective online help, in addition to a user's guide which is distributed as a Word document. This manual lists a number of features slated for versions later than 1.2, as described below. The most useful new feature is the calculation of Tm for primers, along with an algorithm for determining the optimal annealing temperature for the PCR.

New Features of Amplify 2.53 beta

A number of minor alterations in version 2.53 make the interface more user-friendly. These include a two-part primer list window, a "scratch" window for working with formatted text, and an improved help menu. Amplify 2.53 beta can now handle degenerate primers and Ns in the target sequence, both of which are features used in working labs. In the Map window, users can find the exact sequence of an amplified fragment (instead of just its length), while the introduction of colors in the target sequence allows one to easily identify regions of interest. Also, one can format primer matches with specific colors and styles. Other options are available in the Preferences and Set Match Parameters dialog boxes. One can save these settings to serve as the default settings for Amplify.

This reviewer encountered no problems running Amplify 2.53 beta, even though it is still called a beta. As the author of the Amplify home page states, the only significant feature lacking in this version is full documentation.

Running Amplify in Windows with Executor

No other freeware program is comparable to Amplify for Windows platforms. Executor is a commercial Macintosh emulator that allows one to run Macintosh programs on non-Macintosh platforms, including DOS, Windows 3.1, Windows 95/98, Linux, and OS/2. Both Amplify 1.2 and 2.53 beta ran surprisingly well using the Win32 version of Executor in Windows 98. The software retained full functionality, although it did require some tweaking to obtain printer support. This reviewer used the aforementioned solution on a daily basis, in order to design primers for PCR or site-directed mutagenesis (figure 6). Although Executor has a few limitations, speed is not one of them. Visit the ARDI Web site for more information on Executor, or to download a demo version of the program.

Amplify is an essential software program for any research lab designing PCR primers. It does an excellent job of accurately predicting the probability for successful PCR experiments, and saves both time and resources. Although the version 2.53 is still considered a beta, it has proven remarkably stable, and lacks only complete documentation. Amplify is a highly recommended program - software no molecular biology research lab or teaching course should do without.

Amplify runs on any Macintosh, though for this review it was only tested on Systems 7.0, 7.5, 8.0, and 8.1. Under these operating systems, Amplify will run with as little as 4Mb of RAM. It has also been used successfully on a Mac Plus running System 6.x with 1 Mb of RAM, as well as a Mac Classic.

Amplify has been released as freeware, with the copyright retained by the author. It is available from many archives of molecular biology software, including the IUBio Archive, as well as its own Amplify home page. The program must be distributed with all accompanying documentation, but there are few other restrictions.