Since the mismatched primer functions poorly in the first few cycles, the PCR reaction is usually run a few cycles longer than a reaction with correctly matched primers. Many published uses of the Directed Mismatch technique involve allelotyping at the the various RAS genes. The user must either supply a restriction site file or else use the default file. The user is asked for the position in her sequence file of the mutation in question, the identity of the mutant base and the wild type base. The user is also asked for the name of an output file in which the program can list the possibly useful single mismatched sites and the alignment of the recognition sites with the gene sequence.
Output also is sent to the screen.
An inexperienced user should follow these directions. All of your responses to the computer requests are terminated by or . When the program asks for your sequence file, type in filename. When the program asks for the enzyme file, just hit to accept the default restriction file. When the program asks for the name of an output file, invent a name such as mygene.mis which you can later print with the DOS print command or edit with your word processor.