Genotyping accuracy of high-resolution DNA melting instruments.
BACKGROUND
High-resolution DNA melting is a closed-tube method for genotyping and variant scanning that depends on the thermal stability of PCR-generated products. Instruments vary in thermal precision, sample format, melting rates, acquisition, and software. Instrument genotyping accuracy has not been assessed.


METHODS
Each genotype of the single nucleotide variant (SNV) (c.3405-29A>T) of CPS1 (carbamoyl-phosphate synthase 1, mitochondrial) was amplified by PCR in the presence of LCGreen Plus with 4 PCR product lengths. After blinding and genotype randomization, samples were melted in 10 instrument configurations under conditions recommended by the manufacturer. For each configuration and PCR product length, we analyzed 32-96 samples (depending on batch size) with both commercial and custom software. We assessed the accuracy of heterozygote detection and homozygote differentiation of a difficult, nearest-neighbor symmetric, class 4 variant with predicted ΔT(m) of 0.00 °C.


RESULTS
Overall, the heterozygote accuracy was 99.7% (n = 2141), whereas homozygote accuracy was 70.3% (n = 4441). Instruments with single sample detection as opposed to full-plate imaging better distinguished homozygotes (78.1% and 61.8%, respectively, χ(2) P < 0.0005). Custom software improved accuracy over commercial software (P < 0.002), although melting protocols recommended by manufacturers were better than a constant ramp rate of 0.1 °C with an oil overlay. PCR products of 51, 100, 272, and 547 bp had accuracies of 72.3%, 83.1%, 59.8%, and 65.9%, respectively (P < 0.0005).


CONCLUSIONS
High-resolution melting detects heterozygotes with excellent accuracy, but homozygote accuracy is dependent on detection mode, analysis software, and PCR product size, as well as melting temperature differences between, and variation within, homozygotes.
Department of Pathology, University of Utah Medical School, Salt Lake City, UT; current address: Laboratory Center, the Second Hospital of Dalian Medical University, Dalian, China;
Mei
M
Li
Department of Pathology, University of Utah Medical School, Salt Lake City, UT;
Luming
L
Zhou
Department of Mathematics, Utah Valley University, Orem, UT.
Robert A
RA
Palais
Department of Pathology, University of Utah Medical School, Salt Lake City, UT; carl.wittwer@path.utah.edu.
Carl T
CT
Wittwer
D004247:DNA / Q000737:chemistry / Q000235:genetics*; D005838:Genotype; D060005:Genotyping Techniques; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics
D004247:DNA