title: Base-pair neutral homozygotes can be discriminated by calibrated high-resolution melting of small amplicons.
abstract: Genotyping by high-resolution melting analysis of small amplicons is homogeneous and simple. However, this approach can be limited by physical and chemical components of the system that contribute to intersample melting variation. It is challenging for this method to distinguish homozygous G::C from C::G or A::T from T::A base-pair neutral variants, which comprise approximately 16% of all human single nucleotide polymorphisms (SNPs). We used internal oligonucleotide calibrators and custom analysis software to improve small amplicon (42-86 bp) genotyping on the LightScanner. Three G/C (PAH c.1155C>G, CHK2 c.1-3850G>C and candidate gene BX647987 c.261+22,290C>G) and three T/A (CPS1 c.3405-29A>T, OTC c.299-8T>A and MSH2 c.1511-9A>T) human single nucleotide variants were analyzed. Calibration improved homozygote genotyping accuracy from 91.7 to 99.7% across 1105 amplicons from 141 samples for five of the six targets. The average T(m) standard deviations of these targets decreased from 0.067 degrees C before calibration to 0.022 degrees C after calibration. We were unable to generate a small amplicon that could discriminate the BX647987 c.261+22,290C>G (rs1869458) SNP, despite reducing standard deviations from 0.086 degrees C to 0.032 degrees C. Two of the sites contained symmetric nearest neighbors adjacent to the SNPs. Unexpectedly, we were able to distinguish these homozygotes by T(m) even though current nearest neighbor models predict that the two homozygous alleles would be identical.
authors.affiliation: Idaho Technology Inc., 390 Wakara Way and Department of Pathology, University of Utah School of Medicine, 50 North Medical Drive 5B426, Salt Lake City, Utah 84108, USA.
authors.forename: Cameron N
authors.initials: CN
authors.lastname: Gundry
authors.forename: Steven F
authors.initials: SF
authors.lastname: Dobrowolski
authors.forename: Y Ranae
authors.initials: YR
authors.lastname: Martin
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Robbins
authors.forename: Lyle M
authors.initials: LM
authors.lastname: Nay
authors.forename: Nathan
authors.initials: N
authors.lastname: Boyd
authors.forename: Thomas
authors.initials: T
authors.lastname: Coyne
authors.forename: Mikeal D
authors.initials: MD
authors.lastname: Wall
authors.forename: Carl T
authors.initials: CT
authors.lastname: Wittwer
authors.forename: David H-F
authors.initials: DH
authors.lastname: Teng
mesh_terms: D020029:Base Pairing; D002138:Calibration; D005838:Genotype; D006720:Homozygote; D006801:Humans; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards
chemical_list: D009841:Oligonucleotides