ROCK: A Spreadsheet-Based Program for the Generation and Analysis of Random Oligonucleotide Primers Used in PCR

Abstract

Oligonucleotide primers used to amplify target DNA regions via PCR should meet certain design criteria to maximize the potential for efficient priming. The Random Oligonucleotide Construction Kit (ROCK), a spreadsheet-based program that runs under Microsoft® Excel® 97 or later version for Microsoft Windows™, was developed to facilitate the design of efficient random oligonucleotide primers. Primer sequences are generated that meet user-defined criteria with regard to G+C content, size of a 3′ GC clamp, maximum intramolecular/intermolecular complementation potential, and maximum intersequence similarity. The user can analyze the intramolecular/intermolecular complementation potential of program-generated primer sequences or of sequences entered manually. The latter may contain any of the standard nucleotide symbols, including ambiguous bases. Primer sequence length, GC%, individual base composition, molecular weight, approximate melting temperature, and mass/volume/concentration relationships can be determined for any sequence generated by ROCK or entered manually.