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

Abstract

We have designed, tested, and validated synthetic DNA molecules that may be used as reference standard controls in the simultaneous detection of mutations in one or more genes. These controls consist of a mixture of oligonucleotides (100 to 120 bases long) each designed for the detection of one or more disease-causing mutation(s), depending on the proximity of the mutations to one another. Each control molecule is identical to 80 to 100 bases that span the targeted mutations. In addition, each oligonucleotide is tagged at the 5' and 3' ends with distinct nucleic acid sequences that allow for the design of complementary primers for polymerase chain reaction amplification. We designed the tags to amplify control molecules comprising 32 CFTR mutations, including the American College of Medical Genetics minimum carrier screening panel of 23, with one pair of primers in a single tube. We tested the performance of these controls on many platforms including the Applied Biosystems/Celera oligonucleotide ligation assay and the Tm Bioscience Tag-It platforms. All 32 mutations were detected consistently. This simple methodology allows for maximum flexibility and rapid implementation. It has not escaped our notice that the design of these molecules makes possible the production of similar controls for virtually any mutation or sequence of interest.

Development of Genomic Reference Materials for Cystic Fibrosis Genetic Testing

“…Three companies have developed synthetic DNA controls for CF testing; Molecular Controls, Inc., 27 Maine Molecular Quality Control, Inc. (Food and Drug Administration-cleared) (Maine Molecular Quality Controls, Scarborough, ME) and AcroMetrix (Benicia, CA) manufacture control materials that cover all 23 of the ACMG/ACOG recommended alleles as well as other commonly assayed alleles. Synthetic controls are useful because they contain multiple alleles in a single sample, and thus provide efficient and cost saving quality control materials.…”

A universal carrier test for the long tail of Mendelian disease

“…Reference samples representing 131 variants were obtained from the Coriell cell repository, a biobank of genomic DNA reference materials. Because patient samples were not available for all targeted mutations, synthetic patient samples were created which contained each mutant sequence of interest (Berwouts et al, 2008;Christensen et al, 2007;Jarvis et al, 2005) as recommended by the Food and Drug Administration's ''Guidance on Pharmacogenetic Tests and Genetic Tests for Heritable A multistage approach was used to design the universal carrier test. Beginning with 2096 probes to assay a large number of disease-causing variants, a series of increasingly stringent quality-control metrics was then applied to obtain the final set of 925 probes for the assay.…”

“…NA10838; Coriell Institute) in an approximately equimolar ratio. A dilution series of plasmid in genomic DNA was used to set the final concentration of plasmid to achieve a synthetic heterozygote genotype in the combined samples (Berwouts et al, 2008;Christensen et al, 2007;FDA, 2007;Jarvis et al, 2005). To maximize efficiency, pools of non-interfering synthetic controls were created, such that each pool contained multiple mutant sequences.…”

“…The universal carrier test was exhaustively validated on a combination of reference genomic DNA samples (Coriell Institute for Medical Research, 2010), synthetic control samples (Berwouts et al, 2008;Christensen et al, 2007;Jarvis et al, 2005) and clinical DNA samples. In brief, extremely high sensitivity, specificity and positive predictive values were achieved by combining multiple redundant probes, a triage strategy ( Table 1) and two-stage follow-up testing for positive carrier couples.…”

Evaluation and use of a synthetic quality control material, included in the European external quality assessment scheme for cystic fibrosis

“…In the United States, the CDC funded projects to promote the development and distribution of control materials [Grody, 2003;Bernacki et al, 2003;Jarvis et al, 2005] and was involved in the Genetic Testing Reference Material Coordination Program [Chen et al, 2005] (GeT-RM; www.phppo.cdc.gov/dls/genetics/qcmaterials/default.aspx). Three commercial entities in the United States, Molecular Controls [Christensen et al, 2007], AcroMetrix [Huang and Pan, 2007], and Maine Molecular Quality Controls, Inc. (MMQCI) [Johnson et al, 2007], have developed synthetic reference materials (RM) for molecular testing. These synthetic RM were developed in different ways and optimized to work on diverse testing platforms.…”