title: Allele dropout caused by a non-primer-site SNV affecting PCR amplification--a call for next-generation primer design algorithm.
abstract: BACKGROUND
  PCR-based technology is indispensable for genetic diagnosis. On the other hand, allele dropout is one significant cause of genotyping errors. Most allele dropout mechanisms are related to annealing failure caused by single nucleotide variant (SNV) situated inside the primer sequences. Here, we demonstrate a novel allele dropout mechanism caused by a non-primer-binding-site SNV.
  
  
  METHODS
  We demonstrate that the apparent homozygosity of NM_000137.1(FAH):c.1035_1037del was caused by allele dropout.
  
  
  RESULTS
  The non-primer-binding-site SNV causes a strong secondary hairpin structure formation of the PCR products and leads to amplification failure. SNV check of the primer sequences per se during primer design is not adequate to avoid allele dropout.
  
  
  CONCLUSIONS
  The next-generation primer design software should analyze the secondary structure of primers and template sequence taking SNV in both sequences into account in order to avoid genotyping errors.
authors.affiliation: Department of Pathology, The University of Hong Kong, Queen Mary Hospital, Hong Kong, SAR, China. ching-wanlam@hk.hk
authors.forename: Ching-wan
authors.initials: CW
authors.lastname: Lam
authors.forename: Chloe Miu
authors.initials: CM
authors.lastname: Mak
mesh_terms: D000465:Algorithms; D000483:Alleles; D016477:Artifacts; D017931:DNA Primers; D005838:Genotype; D060005:Genotyping Techniques / Q000592:standards*; D006720:Homozygote; D006801:Humans; D006867:Hydrolases / Q000235:genetics*; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000592:standards*; D020641:Polymorphism, Single Nucleotide; D012984:Software; D020176:Tyrosinemias / Q000175:diagnosis / Q000235:genetics*
chemical_list: D017931:DNA Primers; D006867:Hydrolases; C020156:fumarylacetoacetase