Quantitative 1-Schritt-DNA-Methylierungsanalyse aus genomischer DNA
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1 Quantitative 1-Schritt-DNA-Methylierungsanalyse aus genomischer DNA Molekulare Diagnostik 2011 Departement Klinische Forschung Abteilung für Humangenetik Experimentelle Hämatologie DKF und Labor für Molekulare Diagnostik
2 Overview Overview Quantitative 1-step DNA methylation analysis Precision and reliability of quantitative PCR Other applications of digest-qpcr
3 Overview Overview Quantitative 1-step DNA methylation analysis Precision and reliability of quantitative PCR Other applications of digest-qpcr
4 Quantitative 1-step DNA methylation analysis DNA methylation Addition of methyl group to cytosine by DNA methyltransferases Functions Genome defense (e.g. silencing of retrotransposons) Genome integrity (homologous recombination between methylated repeats is prevented) Gene silencing / gene regulation - X chromosome inactivation - Parental imprinting - General gene expression regulation (methylation of CpG islands, but also of transcription factor binding sites)
5 Quantitative 1-step DNA methylation analysis DNA methylation analysis: conventional approach Bisulfite treatment of native DNA Native DNA C converted to U Me C remains Me C Any base can be analyzed Multistep procedure Incomplete conversion PCR Detection of conversion
6 Quantitative 1-step DNA methylation analysis DNA methylation analysis: novel approach Methylation-sensitive endonucleases combined with quantitative PCR gdna HpaII FastDigest Unmethylated CCpGG cut Sham digest No endonuclease qpcr qpcr ΔC q % cut molecules = % unmethylated molecules
7 Quantitative 1-step DNA methylation analysis 1-step DNA methylation analysis Methylation-sensitive endonucleases combined with qpcr gdna HpaII FastDigest Unmethylated CCpGG cut qpcr 1 STEP? Sham digest No endonuclease qpcr ΔC q Reaction mix: DNA, MS-endonuclease (FastDigest), qpcr reagents Reaction conditions: 10 min at 37 C, then qpcr >>> Would be the first assay for 1-step DNA methylation analysis
8 Quantitative 1-step DNA methylation analysis SNRPN, PWS and AS SNRPN, an imprinting locus: - Maternal allele methylated - Paternal allele unmethylated - Expected methylation level 50% SNRPN deletion or uniparental disomy Prader-Willi syndrome (PWS) - only maternal allele present - SNRPN 100% methylated Angelman syndrome (AS) - only paternal allele present - SNRPN 0% methylated
9 Quantitative 1-step DNA methylation analysis First test: mix of PWS and AS DNA, measure SNRPN Disillusioning
10 Precision and reliability of quantitative PCR Imprecision due to digest or qpcr? Technical replicates: Imprecision is due to qpcr qpcr: Used for a multitude of applications, including diagnostic tests requiring highest reliability >>> Which factors affect qpcr precision? >>> How can reliability of qpcr be maximized? >>> Investigation of two common qpcr devices
11 Overview Overview Quantitative 1-step DNA methylation analysis Precision and reliability of quantitative PCR Other applications of digest-qpcr
12 Improving precision and reliability of quantitative PCR Capillary-based LightCyclers Standard conditions Technical replicates with SNRPN assay C q differences would indicate a deletion in a diagnostic setup
13 Precision and reliability of quantitative PCR Capillary-based LightCyclers All carousel positions filled Standard conditions Complete filling of carousel reduces temperature inhomogeneity in thermal chamber Wilhelm et al. (ClinChem 2000): Such effects are due to incomplete template DNA denaturation
14 Improving precision and reliability of quantitative PCR Capillary-based LightCyclers All carousel positions filled Standard conditions Carousel filled, 5 seconds denaturation Increase denaturation from 0 to 5 seconds >>> complete carousel rotation in thermal chamber >>> thermal differences leveled off >>> Simple measures dramatically increase precision and robustness of qpcr (>>> von Kanel et al., ClinChem 2007)
15 Precision and reliability of quantitative PCR LightCycler 480 (plate-based qpcr instrument) Temperature inhomogeneities have previously been reported SNRPN assay on LightCycler 480: positional effects detectable Conditions as recommended by supplier
16 Precision and reliability of quantitative PCR LightCycler 480 (plate-based qpcr instrument) Temperature inhomogeneities have previously been reported SNRPN assay on LightCycler 480: positional effects detectable Increasing initial denaturation to 5 minutes: helps Conditions as recommended by supplier Increased denaturation time
17 Precision and reliability of quantitative PCR LightCycler 480 (plate-based qpcr instrument) Temperature inhomogeneities have previously been reported SNRPN assay on LightCycler 480: positional effects detectable Increasing initial denaturation to 5 minutes: helps Adding DMSO to reaction: as precise as capillary-based LightCycler Conditions as recommended by supplier Increased denaturation, and addition of DMSO Increased denaturation time
18 Precision and reliability of quantitative PCR Additional findings of qpcr precision study Development of statistical tests allowing detection of positional effects in everyday reaction setups >>> Non-parametric: Based on C q -increase / decrease within neighboring replicates >>> Semi-parametric: Based on correlations between well temperatures (assessed with melting curve analysis) and C q s within replicates Besides from DNA denaturation, primer binding is critical for precision in qpcr >>> avoid over-optimization Detecting and resolving position-dependent temperature effects in real-time quantitative PCR von Kanel T, Gerber D, Wittwer CT, Hermann M, Gallati S
19 Overview Overview Quantitative 1-step DNA methylation analysis Precision and reliability of quantitative PCR Other applications of digest-qpcr
20 Quantitative 1-step DNA methylation analysis DNA methylation analysis functioning Linear response when mixing PWS and AS Inclusion of an unmethylated control locus in reaction setup allows......correction for incomplete digestion >>> low-quality DNA analyzable...simultaneous assessment of the copy number status of SNRPN
21 Quantitative 1-step DNA methylation analysis Blinded analysis 35 PWS / AS patients and normal controls All individuals correctly diagnosed by assessing SNRPN methylation >>> Assay applicable in diagnostics SNRPN methylation measured (%) Angelman syndrome normal Prader-Willi syndrome
22 Facilitation of quantitative DNA methylation analysis Summary qosma Quantitative one-step DNA methylation analysis (qosma) is closed-tube requires little hands-on is well-suited for high throughput analysis allows simultaneous copy number assessment Possible drawbacks are limited number of analysable sites incomplete digestion (>>> not for residual disease etc.)
23 Quantitative 1-step DNA methylation analysis Summary qosma Quantitative one-step DNA methylation analysis (qosma) is closed-tube requires little hands-on is well-suited for high throughput analysis allows simultaneous copy number assessment Possible drawbacks are limited number of analysable sites incomplete digestion (>>> not for residual disease etc.)
24 Quantitative 1-step DNA methylation analysis
25 Overview Overview Quantitative 1-step DNA methylation analysis Precision and reliability of quantitative PCR Other applications of digest-qpcr
26 Other applications of digest-qpcr Other applications of digest-qpcr Usage is not limited to methylation-sensitive endonucleases: - Genotyping: alternative to RFLP - Cancer diagnostics: reduction of wildtype signals in allele-specific qpcr
27 Other applications of digest-qpcr Systemic mastocytosis (SM) Abnormal proliferation and accumulation of mast cells >>> fatigue, skin lesions (urticaria pigmentosa), etc. Clinical course: from no adverse effects to deterioration within months Most SM patients: 2447 AT point variation (D816V) in exon 17 of KIT gene >>> constitutive activation of KIT, and resistance to imatinib Detection of D816V: should be sensitive as possible >>> allele-specific qpcr
28 Other applications of digest-qpcr Allele-specific quantitative PCR (AS-qPCR) Goal: - Amplify as much of the mutated alleles as possible > sensitivity - Amplify as little of the wildtype alleles as possible > specificity D816V: Forward primer is specific for mutated allele but amplification of wildtype allele is not fully suppressed Tricks for increased specificity: - Insert additional mismatches in mutation-specific primer >>> might reduce sensitivity of assay - Add blocked primer that is specific for wildtype allele >>> might require extensive optimization - Add endonuclease that cuts wildtype allele?
29 Digest-qPCR applied to KIT D816V KIT D816V: Alw26I cuts wildtype allele Compare AS-qPCR with and without Alw26I Mix Without endonuclease With endonuclease Alw26I HMC-1 Mix 1: HMC-1 Mix 1: HMC-1 Mix 1:1' HMC-1 Mix 1:10' Negative controls (n = 2) (n = 22) >>> Substantial higher C q in negative controls >>> Increased specificity
30 Digest-qPCR applied to JAK2 V617F JAK2 V617F: - Associated with chronic myelogenous leukemia (CML) - BsaXI cuts wildtype allele; not available as FastDigest Compare AS-qPCR with and without BsaXI; 30 min incubation
31 Digest-qPCR applied to JAK2 V617F JAK2 V617F: - Associated with chronic myelogenous leukemia (CML) without BsaXI - BsaXI cuts wildtype allele; not available as FastDigest with BsaXI Compare AS-qPCR with and without BsaXI; 30 min incubation
32 Take-home messages Take-home messages qpcr assays targeting genomic DNA might be prone to incomplete target denaturation >>> positional effects If assays are overly optimized >>> primers might be prone to incomplete binding >>> positional effects Endonuclease digestion of genomic DNA and quantitative PCR can be combined in a single reaction Reaction principle has many potential applications: DNA methylation analysis, genotyping, cancer diagnostics, etc. The digest is quantitative, but does usually not reach 100%
33 Thanks Thanks to Division of Human Genetics, Bern: - Sabina Gallati and Dominik Gerber - Franziska Gisler, Christopher Jackson, and André Schaller, - Lab staff Experimental Hematology and Molecular Diagnostics, Bern: - Elisabeth Oppliger Leibundgut, Naomi Porret, and Barbara Hügli - Lab staff ARUP lab, Utah: - Carl Wittwer and Mark Hermann Institute for Medical Genetics, Zurich: - Alessandra Baumer and Eva Wey
34 Thanks Thanks to Division of Human Genetics, Bern: - Sabina Gallati and Dominik Gerber - Franziska Gisler, Christopher Jackson, and André Schaller, - Lab staff Experimental Hematology and Molecular Diagnostics, Bern: - Elisabeth Oppliger Leibundgut, Naomi Porret, and Barbara Hügli - Lab staff ARUP lab, Utah: - Carl Wittwer and Mark Hermann Institute for Medical Genetics, Zurich: YOU - Alessandra Baumer and Eva Wey
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