Dr. Davide Barbagallo P.h.D. Università di Catania Dip. Sc. Biomediche Sez. di Biologia Generale, Biologia Cellulare, Genetica Molecolare G.

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1 High Resolution Melting (HRM) Analysis Dr. Davide Barbagallo P.h.D. Università di Catania Dip. Sc. Biomediche Sez. di Biologia Generale, Biologia Cellulare, Genetica Molecolare G. Sichel Unità di BioMedicina Genomica e dei Sistemi Complessi, Genetica, BioInformatica

2 PCR: a typical cycle 1) Denaturation (94 C) 2) Annealing ( x C) 3) Extension (72 C or other) PCR: the main actors 1) Template (DNA) 2) Buffer (Mg++) 3) Primers 4) dntp 5) Enzyme (Taq polymerase) 6) Thermal cycler

3 PCR

4 PCR PHASES IN LINEAR VIEW In fase esponenziale la funzione rappresentativa della curva è: Progressione geometrica di ragione 2 e fattore di scala 1, cioè F(x) = 2 n, con n= numero di cicli

5 REAL-TIME PCR: THE ENTIRE PROCESS OF PCR IN REAL TIME Real-time PCR is the continuous collection of fluorescent signal from one or more polymerase chain reactions over a range of cycles. From Real Time PCR, edited by M T Dorak, 2007.

6 SYBR Green: an example of incorporation of a free dye into the newly formed double-stranded DNA product DNA-dye complex results in a dramatic increase in fluorescence output of roughly 2,000 times the initial, unbound, fluorescent signal Pros: 1) Low cost; 2) Ease of assay development; 3) The same detection mechanism can be used for every assay Cons: 1) Not specificity; 2) Toxic for reaction; 3) No Multiplex reaction

7 SYBR Green NEEDS DISSOCIATION CURVE The PCR product Tm is 87.5 C (curves indicated by +). Complete absence of primer-dimer is rarely achieved in the PCR negative control (curve indicated by ). As seen in this example, 1 out of 3 negative triplicates shows dimers (with a Tm of 78.5 C). The two sets of curves are usually clearly separated with a 10 C shift between Tm of primer-dimers and the specific PCR product. drfu/dt

8 HRM Analysis: main features HRM analysis differs from standard melt curve analysis in three ways: 1. Chemistry HRM analysis uses brighter dyes at higher concentrations 2. Instruments HRM analysis requires instruments that collect fluorescence data at finer temperature resolution 3. Software HRM analysis requires more sophisticated software which uses new fluorescent scaling algorithms and plots

9 HRM Analysis: what is it? High Resolution Melting (HRM) analysis is a new, post-pcr analysis method that could be used for identifying genetic variation in nucleic acid sequences. Simple and fast, this method is based on PCR melting (dissociation) curve techniques and is enabled by the recent availability of improved double-stranded DNA (dsdna) binding dyes along with next-generation real-time PCR instrumentation and analysis software. HRM analysis can discriminate DNA sequences based on their composition, length, GC content, or strand complementarity

10 HRM Analysis: how does it function? HRM analysis starts with PCR amplification of the region of interest in the presence of a dsdna binding dye. This binding dye has a high fluorescence when bound to dsdna and low fluorescence in the unbound state. Amplification is followed by a high resolution melting step using instrumentation capable of capturing a large number of fluorescent data points per change in temperature, with high precision. When the dsdna dissociates (or melts) into single strands, the dye is released, causing a change in fluorescence. The result is a melt curve profile characteristic of the amplicon.

11 Saturating but not toxic activity of MeltDoctor dye Vs. SYBR Green dye

12 DNA melt profile

13 HRM Analysis Workflow

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15 The resulting profiles can provide valuable information for mutation screening, genotyping, methylation, and other investigative applications.

16 SNP Genotyping When designing primers for HRM SNP genotyping analysis, the amplicon length should be short to avoid the detection of SNP variants outside of the region of interest. However, smaller amplicons may produce lower fluorescence signals, presumably due to less dye incorporation in the shorter sequence. In order to ensure adequate dye fluorescence signals, the amplicon length should be approximately bp including primers (target sequence length of 30 bp in addition to two 25 bp primer sequences).

17 Homozygous Vs. Heterozygous

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19 HRM analysis uses two profile observations: 1. Melt curves that are similar in shape but that are distinguishable from each other by difference in melting temperature (Tm) of the amplicon. Typically such profiles are generated by homozygous variant samples that are being compared to a wild type sample. In such situations, the Tm difference between samples is due to sequence variation from the wild type. 2. Melt curves displaying a distinct curve shape from homozygote melt curves. These profiles are usually due to the presence of base pairing mismatches (heteroduplexes) present in the PCR product mix.

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21 Mutation Screening When using HRM analysis to screen for unknown mutations, the entire gene length needs to be screened, and the amplicon length used can be long, typically 300 bp. The genomic region of interest should be divided into amplicons with lengths <250 bp, which will produce 300 bp amplicon sequences when 25-mer forward and reverse primers are used. Target sequences should overlap by at least 25 bp to ensure complete coverage of the region of interest.

22 Epigenetica Si intende per Epigenetica una qualunque attività di regolazione dei geni tramite processi chimici che non comportino cambiamenti nel codice del DNA ma possono modificare il fenotipo dell individuo e/o della progenie. Ad esempio: - metilazione del DNA; - acetilazione degli istoni. Questi fenomeni epigenetici alterano l'accessibilita' fisica al genoma da parte di complessi molecolari deputati all'espressione genica e, quindi, alterano il grado di funzionamento dei geni.

23 La metilazione del DNA nei vertebrati avviene tipicamente nei siti CpG (citosina-fosfato-guanina; che si ha ove la citosina è direttamente seguita da una guanina nella sequenza del DNA); tale metilazione risulta nella conversione della citosina in 5-metilcitosina. La formazione del Me-CpG è catalizzato dall'enzima DNA metiltransferasi. I siti CpG sono poco comuni nel genoma degli invertebrati mentre sono spesso trovati con maggior densità nei promoters genici dei vertebrati, in cui sono collettivamente denominati isole CpG. Lo stato di metilazione di questi siti CpG può avere un grave impatto sull'attività/espressione genica.

24 Imprinting genomico

25 Alcuni geni vengono espressi solo se sono stati trasmessi dal padre, mentre altri vengono espressi solo se vengono trasmessi dalla madre. Il fenomeno della espressione differenziale dipendente dal sesso del genitore di origine viene chiamato imprinting. Mentre nella sindrome di Praeder-Willi è il cromosoma 15 materno ad essere presente in duplice copia, nella sindrome di Angelmann abbiamo una duplice copia del cromosoma paterno (si parla di Unisomia Diparentale). In particolare in entrambe le sindromi la regione genomica interessata è quella del chr 15q11-q13. Nella PWS tale regione è metilata nel chr di origine materna e, siccome gli alleli paterni vengono persi per delezione, i geni contenuti nella controparte materna, essendo metilati, non possono esprimersi. Viceversa, nella AS sono i chr 15 paterni ad essere in duplice copia, con conseguente iperattività di quella coppia di cromosomi.

26 Lo stato complessivo di metilazione in una cellula può anche essere un fattore accelerante nella carcinogenesi come suggerisce l'evidenza che l'ipometilazione genomica può portare ad instabilità cromosomica e crescenti tassi di mutazione. Lo stato di metilazione di alcuni geni può essere usata come biomarker per la tumorigenesi. Per esempio, l'ipermetilazione del pi-class glutathione S- transferase gene (GSTP1) appare come un affidabile indicatore del tumore alla prostata.

27 Methylation-sensitive (MS) HRM MS-HRM followed by Sanger-based DNA sequencing is a fast, simple method for methylation studies that can be manipulated to provide sensivity for methylation levels as low as 0.1 2%.

28 Step 1: bisulfite conversion Genomic DNA is treated with bisulfite to convert unmethylated cytosines to uracil through deamination. The same treatment does not affect methylated cytosines!

29 Step 2: primers design and PCR PCR primer design for MS-HRM is critical the the free online tool from Applied Biosystems, Methyl Primer Express Software v1.0, was developed specifically for primer design in methylation studies. Successful MS-HRM assays have been published with bp amplicons and up to 16 methylation sites.

30 Step 3: optimization of the reaction The goal of optimization is to identify the best PCR primer set and PCR cycling conditions for the differentially methylated region of interest. It is good practice to evaluate PCR reactions by real-time PCR, MS-HRM, and electrophoresis on high- percentage agarose gels.

31 Step 4: MS-HRM Standard Curve and Data Analysis MS-HRM analysis is based on comparing melt profiles of experimental samples to profiles from DNA with known methylation levels. Universally (or 100%) methylated DNA is commercially available.

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33 Step 5: Sequencing the HRM Product The reaction products can then be sequenced to validate results and identify exact patterns of methylation.