Design, Development, Validation, and Use of Synthetic Nucleic Acid Controls for Diagnostic Purposes and Application to Cystic Fibrosis Testing

Todd M. Christensen^*, Mohamed Jama, Victor Ponek, Elaine Lyon, Jean Amos Wilson, Marcy L. Hoffmann^* and Bassem A. Bejjani^*

From the Sacred Heart Medical Center, * Spokane, Washington; ARUP Laboratories, Salt Lake City, Utah; Specialty Laboratories, Incorporated, Santa Monica, California; and Focus Diagnostics, Incorporated, Cypress, California

We have designed, tested, and validated synthetic DNA moleculesthat may be used as reference standard controls in the simultaneousdetection of mutations in one or more genes. These controlsconsist of a mixture of oligonucleotides (100 to 120 bases long)each designed for the detection of one or more disease-causingmutation(s), depending on the proximity of the mutations toone another. Each control molecule is identical to 80 to 100bases that span the targeted mutations. In addition, each oligonucleotideis tagged at the 5' and 3' ends with distinct nucleic acid sequencesthat allow for the design of complementary primers for polymerasechain reaction amplification. We designed the tags to amplifycontrol molecules comprising 32 CFTR mutations, including theAmerican College of Medical Genetics minimum carrier screeningpanel of 23, with one pair of primers in a single tube. We testedthe performance of these controls on many platforms includingthe Applied Biosystems/Celera oligonucleotide ligation assayand the Tm Bioscience Tag-It platforms. All 32 mutations weredetected consistently. This simple methodology allows for maximumflexibility and rapid implementation. It has not escaped ournotice that the design of these molecules makes possible theproduction of similar controls for virtually any mutation orsequence of interest.