Noncontinuously binding loop-out primers for avoiding problematic DNA sequences in PCR and sanger sequencing.

We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.

Validation of an unlabeled probe melting analysis assay combined with high-throughput extractions for genotyping of the most common variants in HFE-associated hereditary hemochromatosis, C282Y, H63D, and S65C.

Hereditary hemochromatosis is an inherited disorder of iron metabolism, characterized by high absorption of iron by the gastrointestinal tract leading to a toxic accumulation of iron in various organs and impaired organ function. Three variants in the HFE gene (p.C282Y, p.H63D, and p.S65C) are commonly associated with the development of the disease. Of these, p.C282Y homozygotes are at the highest risk. Compound heterozygotes of p.C282Y along with p.H63D or p.S65C have reduced penetrance. Furthermore, p.H63D homozygotes are not at an increased risk and little is known about the risk associated with homozygocity for p.S65C. Our current clinical assay for the three common HFE variants utilizes the LightCycler platform and paired probes employing fluorescent resonance energy transfer. To increase throughput and decrease costs, we developed a method whereby automated extraction was combined with unlabeled probes and differential melt profiles to detect these variants using the LightCycler 480 instrument. Using this approach, 43 samples extracted with three different extraction platforms were correctly genotyped. These data demonstrate that the newly developed assay to genotype the HFE mutations p.C282Y, p.H63D, and p.S65C, combined with high-throughput extraction platforms, is accurate and reproducible and represents an alternative to previously described tests.

Simultaneous genotyping of rs12979860 and rs8099917 variants near the IL28B locus associated with HCV clearance and treatment response.

Recent genome-wide association studies have identified two host single-nucleotide polymorphisms (SNPs) near the IL28B gene (rs12979860 C/T and rs8099917 T/G) that are associated with sustained virological response in patients infected with the hepatitis C virus. Herein, we describe a rapid multiplexed dual-color fluorescence resonance energy transfer (FRET) probe assay that accurately genotypes for both SNPs simultaneously. A single-nucleotide extension assay was also developed for verification of genotypes. Agreement (100%) was observed in genotype calls between the FRET and single-nucleotide extension methods for both SNPs, yielding 100% analytical sensitivity and specificity. By using the FRET assay, 443 samples of varying ethnic backgrounds were genotyped and six different compound genotypes (rs12979860/rs8099917) were detected in whites, Asians, Middle Easterners, Hispanics, and African Americans, at the following frequencies: CC/TT (39.2%, 78.9%, 40.0%, 33.9%, and 16.8%), CT/TT (20.8%, 0%, 40%, 9.3%, and 37.0%), TT/TT (2.4%, 0%, 0%, 3.4%, and 35.3%), CT/TG (24.0%, 19.7%, 20%, 39.8%, and 3.4%), TT/TG (8.0%, 1.4%, 0%, 3.4%, and 5.9%), and TT/GG (5.6%, 0%, 0%, 10.2%, and 1.7%), respectively. The multiplexed FRET assay can be used to effectively genotype for both SNPs in a single tube, with high analytical sensitivity and specificity.