Synthesized
Multiple Control DNA Sequences Optimize Quality Control Maintaining highly reliable molecular genetic analyses requires simultaneously testing controls for all tested sequences. Given a DNA and/or RNA specimen with appropriate target sequences from any species or infectious organism, PCR amplification can synthesize any previously reported sequence. These synthesized sequences provide standards to be tested simultaneously with unknown samples to optimize and maintain quality control for all reported mutations and polymorphisms no matter how rare or unavailable. Selected short synthesized mutant or variant sequence primers and flanking primers are used to independently amplify adjacent upstream and adjacent downstream sequences from total genomic DNA. Then the upstream and downstream fragments are amplified together to produce a longer fragment with the variant sequence in the center. Among the synthesized cystic fibrosis gene fragments are (1) all 25 mutations recommended in the core cystic fibrosis panel including four cystic fibrosis mutations previously unavailable through any source (2184delA, 1078delT, 1898+1->G->A, and I148T), (2) four homozygous mutations on one exon 11 fragment, (3) four heterozygous mutations in one exon 11 fragment, and (4) two fragments with 7 mutations from exons 4 and 11 that were spliced together and cloned. Our cystic fibrosis control set amplifies 29 homozygous control mutations in 3 multiplex PCR reactions that are analyzed on 2 Innogenetics test strips. Other applications include amplifying, splicing, and cloning multiple noninfectious sites as a control for multiplex PCR analysis of infectious organisms. Unique fragment patterns based on size and length would distinguish each infectious agent tested by multiplex screening tests. In addition, multiplex controls for the Huntington disease trinucleotide repeat site could include the 33 and 39 repeat fragments which represent the limits of the inconclusive or "gray zone" results that cannot be used to predict patient phenotype reliably. Similarly, defined SSR repeat length control alleles at predetermined sizes like 3-repeat SSR fragment ladders would provide an extremely reliable standard for identity databases. Synthesized multiple SNP and STR alleles will provide optimal controls for other multiplex applications. Our synthesized mutations demonstrate three principles: (1) placing multiple homozygous mutant sequences from adjacent and distant gene locations on the same single cloned control DNA sequence, (2) analyzing multiple homozygous and/or heterozygous controls from different gene locations simultaneously, and (3) using multiplex controls in multiplex assays streamlines quality control to save time and costly test platforms.
Patent applied.