Robustness of single-base extension against mismatches at the site of primer attachment in a clinical assay.
DNA genotyping is important for epidemiological and clinical studies and diagnosis for individuals. Genotyping error can strongly influence the outcome of such investigations. One possible reason for genotyping error is additional DNA sequence variation, which can lead to allelic dropout. Based on a published study where allelic dropout occurred in genotyping the cholesteryl ester transfer protein TaqIB polymorphism by a TaqMan-based method, we investigated the susceptibility of the single-base extension (SBE)-based GenoSNIP method to additional sequence variation at the primer attachment site. SBE genotyping was applied to 147 patient samples with known alleles and to synthetic SBE templates. Variables were positions of nucleotide mismatches, yield of SBE reactions, primer design, and ratio of alleles in the template. No allelic dropout occurred when genotyping the TaqIB polymorphism regardless of the reported nucleotide mismatch. Yields of SBE assays critical for allelic dropout were decreased in the presence of the reported nucleotide mismatch depending on SBE assay design. In a systematic mutation scan, only the position immediately adjacent to the polymorphism caused allelic dropout under standard conditions. Depending on SBE assay design, changes in allelic ratio due to a nucleotide mismatch were similar in appearance to changes due to sample mixture or copy number variation. In conclusion, we found the SBE genotyping assays to be relatively robust against interfering DNA variations. The importance of appropriate design and validation of assays, especially in regard to critical yields and potentially interfering nucleotide mismatches, should be emphasized particularly in clinical settings. Care should be taken when interpreting observed changes in the allelic ratio, which could be caused by nucleotide mismatches, sample mixtures, or copy number variation.
Kirsten
Holger
H
IKIT/BBZ, Faculty of Medicine, Center for Biotechnology and Biomedicine, University of Leipzig, Johannisallee 30, 04103, Leipzig, Germany.
Teupser
Daniel
D
Weissfuss
Jana
J
Wolfram
Grit
G
Emmrich
Frank
F
Ahnert
Peter
P
D000483:Alleles; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D005821:Genetic Techniques; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D019914:Taq Polymerase
D017931:DNA Primers; D019914:Taq Polymerase