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Automated DNA extraction, quantification, dilution, and PCR preparation for genotyping by high-resolution melting
Michael T Seipp1, Mark Herrmann, Carl T Wittwer
1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA. seippmt@aruplab.com
Journal of Biomolecular Techniques : JBT
|December 2, 2010
Summary
High-resolution amplicon melting (HRM) can genotype single nucleotide polymorphisms like H63D, even when melting temperatures are identical. This method uses quantitative heteroduplex analysis and automation for accurate genetic testing.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- High-resolution amplicon melting (HRM) is a genotyping technique using two PCR primers and a DNA dye.
- HRM differentiates genotypes by melting curve shape and temperature (Tm), but struggles with single nucleotide polymorphisms (SNPs) with identical Tm.
- The H63D mutation, linked to hemochromatosis, is an example of such a SNP.
Purpose of the Study:
- To develop and validate a high-resolution amplicon melting (HRM) method for accurate genotyping of the H63D mutation.
- To demonstrate the utility of quantitative heteroduplex analysis for distinguishing homozygous variants with identical melting temperatures.
- To showcase an automated, hands-off integrated solution for DNA extraction, quantification, and PCR preparation for HRM genotyping.
Main Methods:
- Utilized high-resolution amplicon melting (HRM) with a generic, saturating DNA dye.
- Employed quantitative heteroduplex analysis by adding wild-type (WT) DNA to create artificial heterozygotes for distinguishing homozygous variants.
- Integrated automated DNA extraction (using Agencourt® Genfind™ v.2), quantification, dilution, and PCR preparation with a Biomek® NX robot.
- Collected HRM data on a LightScanner-96 and analyzed results using custom software.
Main Results:
- The HRM method, incorporating quantitative heteroduplex analysis, successfully distinguished all genotypes, including homozygous variants of the H63D mutation.
- In a blinded study of 42 H63D samples, 41 genotypes (97.6%) were concordant with dual hybridization probe genotyping.
- A single discordance was attributed to low sample DNA concentration affecting heterozygote detection.
Conclusions:
- Automated DNA extraction and preparation coupled with HRM and quantitative heteroduplex analysis provide a robust solution for genotyping SNPs like H63D.
- Accurate DNA quantification is critical for the reliability of this automated genotyping workflow.
- This integrated approach offers a high-throughput and accurate method for genetic analysis.
