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Modified Allele Dosage (MAD) Genotyping by Hi-Res Melting  of Whole Amplicons

Modified Allele Dosage (MAD) Genotyping by Hi-Res Melting  of Whole Amplicons. Jason T. McKinney Scientist Human Genetics Applications. Modified what? … Who’s MAD?. M odified A llele D osage = Pre-PCR DNA mixing “MAD”? Well, … PHENCODE=ENCODE+GenPhen, HuGENet,LSDB’s, …

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Modified Allele Dosage (MAD) Genotyping by Hi-Res Melting  of Whole Amplicons

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  1. Modified Allele Dosage (MAD) Genotyping by Hi-Res Melting of Whole Amplicons Jason T. McKinney Scientist Human Genetics Applications

  2. Modified what? … Who’s MAD? • Modified Allele Dosage = Pre-PCR DNA mixing • “MAD”? Well, … PHENCODE=ENCODE+GenPhen, HuGENet,LSDB’s, … • Seems as though “MAD” is a MUST • Genotype by Hi-Res Melting (HRM) of whole amplicons • Detection of heteroduplexes • “Core” application of Hi-Res Melting • Force heteroduplex formation in homozygous samples by mixing DNA pre-PCR • Typically done 1:1 volume:volume

  3. Stealing from Peter? • Must not compromise ability to distinguish “true” heterozygous samples • Homozygous “heteroduplexes” = • “intermediate” quantity of heteroduplex molecules relative to “true” heterozygous samples and wild type samples

  4. Ideally …

  5. Optimal Ratio of DNA mixing Palais RA, Liew MA, Wittwer CT. Quantitative heteroduplex analysis for SNP genotyping. Analytical Biochemistry. 2005. In Press.

  6. Optimal ratio, … in English please? • 1X WT DNA + 6X Unknown DNA yields the following heteroduplex ratio’s: • Given HRM detection of ~5-10% variant DNA, these ratio’s should be easily discriminated

  7. Benefits • Genotype sans probe (labeled or unlabeled) • Cost and technical benefit • No asymmetric amplification • Technical benefit • Use “scanning” primers to genotype •  Potential to observe variants other than the SNP of interest • Relative to a probe-based genotyping method

  8. System Requirements • Sequence characterized reference sample (wildtype or variant) • Accurately quantified DNA samples • dsDNA “saturation” dye (LCGreen Plus) • High-resolution instrumentation for melting curve analysis (HR-1, LightScanner) • Well characterized region around SNP is beneficial

  9. Validation of MAD genotyping • Risk burden of HDL haplotypes – 6 genes regulating HDL cholesterol metabolism • Primer design • Area around SNP’s interrogated in 100 chromosomes • Primers designed to avoid observed variants • PCR quality • Primer concentration across annealing temperature gradient • Definitive criteria for Hi-Res Melting success

  10. Methods • Quantify DNA samples (~10ng/l) • Add reference DNA (wildtype) to master mix at 1/7 total DNA • Add unknown DNA at 6/7 total • Amplification – 96 well thermal block • HRM on LightScanner • Automated calling of genotypes

  11. Recipe for a reaction

  12. Cholesterol ester transfer protein (CETP) Hepatic lipase (LIPC) Scavenger receptor protein, beta 1 (SCARB1) IVS7+4 C>T; c1264 A>G; IVS15-30 A>G; c1482+1954 A>C A-763G; C-514T; C-480T; G-250A c1050 C>T GenesSNP’s

  13. Show me the Data!

  14. CETP IVS7 +4 C>T

  15. CETP c1264 A>G

  16. CETP IVS15 –30 A>G

  17. CETP c1482+1954 A>C

  18. LIPC A-763G

  19. LIPC G-250A

  20. SCARB1 c1050 C>T

  21. LIPC C-514T, C-480T

  22. Summary of Data • SNP’s observed • C>T (4), A>G (3), A>C (1), G>A (1) • Fragment size range - 98-261 bp • Strong correlation between fragment size and WT – Het difference (r=0.93) • No correlation between WT – Mut difference and fragment size (r=0.12) • 50-60% GC • 100% genotype concordance • TaqMan (350) • Sequencing (68)

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