Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Jul;9(3):315-9.
doi: 10.2353/jmoldx.2007.060180.

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

Affiliations
Free PMC article

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

Todd M Christensen et al. J Mol Diagn. .
Free PMC article

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.

Figures

Figure 1
Figure 1
Design methodology. A: Each synthetic reference standard is designed to be amplified with a single primer pair that anneals to each primer tag region. Because the oligonucleotides are single stranded, the first round of PCR amplification generates a complementary strand for each synthetic oligonucleotide. B: After this initial cycle, the remaining PCR cycles amplify the DNA fragments in a standard manner. Reference oligonucleotides are designed for each mutation of interest. In some cases, more than one mutation may be incorporated within the same reference oligonucleotide (red circles). C: Assorted mixtures of the various reference oligonucleotides are combined and amplified using a single primer pair to produce synthetic controls carrying one or dozens of mutations that may be visualized in a single reaction tube.
Figure 2
Figure 2
PCR/OLA data. This is a comparison between the synthetic control mixture (A) and a heterozygous ΔF508 genomic control (B) with the Celera Diagnostics CFv3.0 PCR/OLA ASR reagents on an ABI 3100 genetic analyzer.
Figure 3
Figure 3
TAG-IT data. Raw data from the CF32 synthetic control and a wild-type genomic DNA sample are analyzed on the TAG-IT platform using the 40 + 4 CF ASR set. This assay does not generate a F508 wild-type signal, whereas it does produce wild-type signal for G85, G542, and 3905. These discrepancies most likely reflect the distinctiveness of the platforms themselves (ABI’s OLA approach versus Tm Bioscience’s allele-specific primer extension) instead of the performance of the controls.
Figure 4
Figure 4
Reproducibility study. Electropherograms from 100 distinct runs of each of the CF32 control and a wild-type genomic DNA control. These runs are aligned and evaluated within a single Genotyper project file. The aligned electropherograms represent data compiled from 100 consecutive runs performed during standard testing performed at the Sacred Heart Medical Center clinical laboratory throughout the last year.

Similar articles

See all similar articles

Cited by 3 articles

Publication types

Feedback