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? M odified A llele D osage = Pre-PCR DNA mixingM odified A llele D osage = Pre-PCR DNA mixing “MAD”? Well, … PHENCODE=ENCODE+GenPhen, HuGENet,LSDB’s, …“MAD”? Well, … PHENCODE=ENCODE+GenPhen, HuGENet,LSDB’s, … –Seems as though “MAD” is a MUST Genotype by Hi-Res Melting (HRM) of whole ampliconsGenotype by Hi-Res Melting (HRM) of whole amplicons Detection of heteroduplexesDetection of heteroduplexes –“Core” application of Hi-Res Melting Force heteroduplex formation in homozygous samples by mixing DNA pre-PCRForce 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 samplesMust not compromise ability to distinguish “true” heterozygous samples Homozygous “heteroduplexes” =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: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 discriminatedGiven HRM detection of ~5-10% variant DNA, these ratio’s should be easily discriminated

7. Benefits Genotype sans probe (labeled or unlabeled)Genotype sans probe (labeled or unlabeled) –Cost and technical benefit No asymmetric amplificationNo asymmetric amplification –Technical benefit Use “scanning” primers to genotypeUse “scanning” primers to genotype  Potential to observe variants other than the SNP of interest  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)Sequence characterized reference sample (wildtype or variant) Accurately quantified DNA samplesAccurately quantified DNA samples dsDNA “saturation” dye (LCGreen Plus)dsDNA “saturation” dye (LCGreen Plus) High-resolution instrumentation for melting curve analysis (HR-1, LightScanner)High-resolution instrumentation for melting curve analysis (HR-1, LightScanner) Well characterized region around SNP is beneficialWell characterized region around SNP is beneficial

9. Validation of MAD genotyping Risk burden of HDL haplotypes – 6 genes regulating HDL cholesterol metabolismRisk burden of HDL haplotypes – 6 genes regulating HDL cholesterol metabolism 1.Primer design a.Area around SNP’s interrogated in  100 chromosomes b.Primers designed to avoid observed variants 2.PCR quality a.Primer concentration across annealing temperature gradient b.Definitive criteria for Hi-Res Melting success

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

11. Recipe for a reaction

12. Genes SNP’s  Cholesterol ester transfer protein (CETP) Hepatic lipase (LIPC) Hepatic lipase (LIPC) Scavenger receptor protein, beta 1 (SCARB1) 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 A-763G; C-514T; C- 480T; G-250A c1050 C>T c1050 C>T

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 observedSNP’s observed –C>T (4), A>G (3), A>C (1), G>A (1) Fragment size range - 98-261 bpFragment 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% GC50-60% GC 100% genotype concordance100% genotype concordance –TaqMan (350) –Sequencing (68)