Www.roche-applied-science.com High Resolution Melting: History, Technology, and Utility Charles Hardwick, Ph.D Field Applications Consultant HRM - utilized.

High Resolution Melting: History, Technology, and Utility Charles Hardwick, Ph.D Field Applications Consultant HRM - utilized more in Australia and UK than in us maybe cost of sequencing in US vs. other places - want to discuss the history of HRM, the technology required, and the current and potential future uses of HRM

High Resolution Melting What is it?
Melting Curve Analysis is well established as a method to characterize amplicons with SYBR Green I, HybProbe (FRET) or SimpleProbe probes. High resolution melting analysis is an extension of melting curve analysis… enables not only detection of SNPS but also their discovery requires special fluorophores, a high-performance instrument (block homogeneity, suitable filters, optical sensitivity and resolution) and special analysis algorithms. HRM is an extension of the utility of melting curve

History - Background Evolved from need to monitor sequence variation of entire amplicon Single-strand conformation polymorphism Heteroduplex migration Denaturating gel electrophoresis Temperature gradient gel electrophoresis Enzymatic or chemical cleavage Cycle sequencing and gel electrophoresis Denaturing HPLC Mass Spectrophotometry Array analysis Evolved from the need to be able to examine minor sequence varations in large numbers of samples. Other methodologies also used – expensive and slow

History – Melting Curve Analysis
Introduced in 1997 in conjunction with real time PCR With SYBR Green, provides a rough characterization of what product is amplified, and purity of product, indicating specificity of PCR reaction Heterozygote detection possible only with addition of subsequent steps such as amplicon purification and addition of high concentrations or urea With hybridization probes or ‘Simple probes’, can interrogate and detect specific regions of amplicon for sequence alterations Difficult and expensive to screen for unknown mutations due to multiple probes required to span region With a high resolution dye, can detect amplicon and oligonucleotide denaturation, allowing for product identification and SNP detection or discovery in same run. Quantification not possible with HRM Dyes Melting Curve Analysis (MCA) is a well established method to characterize amplicons. This post-PCR technique subjects DNA samples to increasing temperatures and records the details of their dissociation from double-stranded to single-stranded form. The Tm value depends on GC content, length and sequence. In combination with SYBR Green I, melting curve analysis is used for product identification e.g., specific PCR-product vs primer-dimers. For monitoring of small sequence differences (e.g., a SNP) the results are neither robust nor reproducible. In combination with HybProbe (FRET) or SimpleProbe probes, melting curve analysis is used for highly specific and accurate SNP genotyping. However, information is only provided when the sequence variant is covered by the probe. The recently developed High Resolution Melting (HRM) is a refinement of melting curve analysis: It allows to extract even more information, since it identifies the product like with SYBR Green I and additionally detects unknown sequence alterations.

Melting Curve Analysis Established Applications
SYBR Green I for product identification In combination with SYBR Green I, melting curve analysis is used for product identification e.g., specific PCR-product vs primer-dimers. For monitoring of small sequence differences (e.g., a SNP) the results are neither robust nor reproducible.

Melting Curve Analysis Established Applications Melting Curve
SYBR Green I for product identification Fluorescence labeled Probes for Genotyping In combination with HybProbe (FRET) or SimpleProbe probes, melting curve analysis is used for highly specific and accurate SNP genotyping. However, information is only provided when the sequence variant is covered by the probe.

Melting Curve Analysis Established and New Applications
SYBR Green I for product identification High Resolution Melting Dye for Gene Scanning Fluorescence labeled Probes for Genotyping The recently developed High Resolution Melting (HRM) is a refinement of melting curve analysis: It allows to extract even more information, since it identifies the product like with SYBR Green I and additionally detects unknown sequence alterations.

History – High-Resolution Melting
Traditional genotyping methods versus high resolution melting Ideal for screening 1000s of samples for sequence variations Previous gene scanning techniques High throughput Cost Effective Fast Low throughput Expensive Time consuming Evolution of techniques dHPLC Sequencing Real-Time PCR

SNP Discovery and Genotyping Methods
Melting Curve Analysis Summary of what HRM does Previously, from specimen to results required amplification, clean up, matrix loading, then dhplc, etc before sequencing to determine what and where differences were. HRM shortcuts directly to sequencing

Why High Resolution Melting? Robust, non-destructive closed-tube method with many applications; highly informative and flexible. More convenient and cost-effective than current technologies, such as sequencing or dHPLC. ----- Meeting Notes (6/26/12 15:40) ----- Non-destructive means you can clean it up and sequence it.

High Resolution Melting - Technology
Principles Prerequisites Dyes Instrumentation Data Analysis To discuss the Technology required for HRM, we are going to first discuss the principles of HRM, talk about the prerequisites, review the dyes and instrumentation requirements, and what the data look like

High Resolution Melting Amplicon Melting
DNA with PCR heterozygote C + T SNP G A C C C G G Denaturation Increasing reannealing T temperature T G A A + C Intercalating C T fluorescent If after PCR a melting curve is performed with an intercalating but saturating dye like, e.g., LCGreen Plus, several PCR products will combine together as a mixture of perfect or imperfect matches. By raising the temperature, only the real homoduplexes will stay until high temperature is reached. Because of its instability, the heteroduplexes will melt away earlier. A dye A A T T G G

High Resolution Melting
Raw Data This is what raw data looks like

Amplicon Melting Variation in Melting Temperature (Tm)
The Tm of an amplicon depends mainly on GC content. Alterations in the amplicon may influence the Tm. Amplicon Melting of homozygote samples (containing homoduplexes of wildtype or mutant DNA) give very similar curve shapes. Amplicon Melting of heterozygote samples (containing homo and heteroduplexes) give curve shapes which are highly distinct. Highest Stability Lowest Stability G:C > A:T > G:G > G:T = G:A > T:T = A:A > T:C > A:C > C:C Why HRM works; What are the factors influencing melting behaviour? The melting temperature mainly depends on the GC content and the length of the double-stranded DNA. This is because there are three hydrogen bonds between guanine and cytosine, making their binding stronger than between adenine and thymine with only two hydrogen bonds. Low GC content causes a low Tm and high GC content a high Tm.. A genetic alteration may change the Tm of the amplicon, since the exchange itself and the nearest neighbour of the exchange may influence the stability of the nucleic acid. This implies, that sometimes a wildtype and a mutant may not be discriminated, since both will melt in similar fashion (depending on base exchange). The heterozygous sample, which contains homoduplexes of wildtype and mutant and heteroduplexes as well, will melt in a very dissimilar manner. Amplicon melting does not identify specific sequences (i.e., no genotyping). Only differences between two genomes are detected.

Technology - Prerequisites and Innovations What Is Needed to Perform HRM?
Novel intercalating dye to identify heteroduplex DNA saturating, non-inhibitory, ds DNA binding without redistribution during melting Precise Instrument to allow genotyping and/or mutation scanning of whole PCR products. homogenous temperature profile and temperature control high sensitivity optical system (light source, filters and detection system) Flexible Data Analysis Software Sensitive and specific algorithms to distinguish detected differences Easy to use, easy to adjust Melt-standard compatible Touch on points on slide

High Resolution Melting Non-Saturating vs Saturating Dyes
homoduplexes heteroduplexes VS Fluorescent ds-DNA specific dyes (e.g., SYBR Green I) individual curves not sharp overlap is the same for homo- and heteroduplexes vs Saturating dye uniform, sharp signals only sequence but not dye makes a difference Non-saturating dyes don’t bind at every possible base pair in ds DNA ladder, saturating dyes do

High Resolution Melting High Resolution Melting Dye in Action
Non-Saturating Dye- SYBR Green I → No decrease in fluorescence Heat Saturating Dye- LightCycler HRM Master Non-saturating dyes can also exhibit redistribution during melting that eliminates the ability to detect minor differences. → Decrease in fluorescence Heat

High Resolution Melting Dyes
Gundry et al tried a number of common and uncommon dyes for HRM SYBR Green 1 SYBR Gold Ethidium bromide Pico Green TOTO-1 YOYO-1 Requirements: Saturating non-inhibitory to PCR reaction Sufficient fluorescent levels for detection Allows heteroduplex detection Gundry et al were looking for dyes appropriate for HRM and compared these dyes with LCGreen Characteristics required

High Resolution Melting Dyes
Very few dyes meet the requirements LC Green – Idaho Technologies – somewhat inhibitory R27 – Biolight – limited heteroduplex detection EvaGreen – Biotium – somewhat inhibitory, though less than SYBR ResoLight – Roche Signal 7x higher than LC Green No PCR inhibition within 8x concentration range Improved stability over LC Green or R27 Well suited to heteroduplex differentiation Dyes seen in literature and

Prerequisites and Innovations What Is Needed to Perform Hi Res Melt?
Precise Instrument to allow genotyping and/or mutation scanning of whole PCR products. homogenous temperature profile and temperature control high sensitivity optical system (light source, filters and detection system) Instruments requirements – need exactly same PCR reaction in each well to be sure that target differences are not masked by instrument-induced variations, and need instrument that has capabilities to detect the differences

LightCycler® 480 System Thermocycler
Six Peltier elements: semi-conductors where direction of current either cools or heats the thermoblock. Therma-BaseTM Cooling body Heat pumps MWP Mount Heated lid Includes Therma-BaseTM for optimized heat exchange which results in excellent overall temperature homogeneity. Allows to finish a PCR run: 96 wells in < 1 hour 384 wells in < 40 min. When a multiwell plate is inserted it is carefully positioned on the mount which provides a perfect fit. Heating and cooling is done by peltier elements. These are semiconductors sandwiched between two ceramic plates and work as electronic heat pumps. Their function can be switched to cooling by reversing the current. But it is underneath the peltier elements that we see something very unique and plays a very important role for optimized heat transport to and from the plate: it is called a therma base. The combination of the peltier and therma base allows for a PCR run for 384 samples to be finished in 40 minutes. New technology for thermocyclers Unique to LC480

Thermal Uniformity Intra-Run Reproducibility of 96 Replicates
Positions: A1-O23 Positions: B1-P23 Positions: A2-O24 These are the results for three different target temperatures on the LightCycler® 480 Instrument.

LightCycler® 480 Performance Absolute Quantification, SYBR Green I
2-step RT-PCR Target: h-HPRT Example of absolute quantification with SYBR green. Simply point out the range of template used 100ng down to 10pg The next thing to point out is the very low standard deviation between replicates. Also comment that the RNA was reverse transcribed with reverse transcriptase..

LightCycler® 480 Instrument
Thermal Uniformity Instrument Comparison -96 wells LightCycler® 480 Instrument Standard Instrument Here, the variation of the measured Tm from the expected Tm of the oligonucleotide was graphed for all 96 wells, using the expected Tm as zero. The result was compared to a standard instrument (right).

LightCycler® 480 Instrument
Thermal Uniformity Instrument Comparison – 384 wells LightCycler® 480 Instrument Standard Instrument Here, the variation of the measured Tm from the expected Tm of the oligonucleotide was graphed for all 384 wells, using the expected Tm as zero. The result was compared to a standard instrument (right).

LightCycler® 480 Instrument Optical System - Lightpath
Folded optical path to reduce height CCD Camera Optics Filter Filter Lamp Unit Optics cable Optics CCD Camera Lamp Unit To incorporate the long optical path into the LC480, a series of mirrors is used to “bend” the path. This ensures our distance of 80cm is still maintained however is confined to a smaller area. Reference Channel Heated Cover Heated Cover Micro well plate Micro well plate

LightCycler® 480 Optical System Sensitivity and Homogeneity
Xenon lamp CCD camera Five excitation filters Six detection filters Optimized arrangement of optical components Homogeneous excitation and fluorescence detection Filter wheels CCD Camera Multiwell plate Field lens Xenon lamp (sensitivity for all formats – even HybProbe probes!) CCD (instead of photohybrids used in capillary-based LightCyclers) Five (5) excitation channels Six (6) detection channels Optimized arrangement of optical components Homogeneous excitation and fluorescence detection Background information: Lifetime of a Xenon lamp: 500 – 1000 h Halogen lamp: 2000 h Argon-ion laser: 5000 – h

LightCycler® 480 Instrument Optical Properties
Light source: high intensity xenon lamp Highest intensity of light over a broad electromagnetic spectrum Degrades (ages) in linearly, without spectral shift. Excitation filters 450, 483, 523, 558, 615 nm The light source for the LC480 is a Xenon lamp. This was chosen because it gives the highest intensity of light over a broad spectrum. Therefore allows for multiplexing of up to 6 channels. Emission filters 500, 533, 568, 610, 640, 670 nm

LightCycler® 480 System Assay Formats and Dyes

External evaluation ARUP (Salt Lake City) study of hardware features
Herrmann, M. G. et al. (2007). "Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping." Clin Chem; June 2007 Comparison between LightScanner assay and LC480 assay shows that the LC480 assay discriminates much better between the different mutations. Heterozygote scanning: LightCycler® 480 equals LightScanner LightCycler® 480 advantages: data density, signal-to-noise ratio, melting rate, speed

High Resolution Melting Software and Data Analysis
Wittwer et al (2003) demonstrated a useful and robust analysis methodology that has the capability to reveal both homo- and hetero-duplex DS DNA configurations Utilizes fluoresence normalization, temperature shift adjustment, and derivative melting curve plots Can reveal extremely minor differences in DS DNA melting curve shape Allows for comparison and adjustment to use melting standards for genotyping Newest software uses same basic analyses proposed by Wittwer in 2003; extremely sensitive and robust; allows use of standards

High Resolution Melting Data Analysis
Raw Data Difference Plot Normalized, Tm-shifted Difference Plot Temperature Shift Normalization The amplicon melting works via several calculation steps, the software will automatically perform: First a normalization of the raw data to 100% (at the beginning) and 0% (at the end of the melting) and then a temperature shift to allow comparison of the curves. The picture in the Difference Plot reveals the required information. Both homoduplexes (blue and green) have very similar curve shapes, which do not show high peaks. Only the heteroduplexes are different (red curve).

Wt/Homo/Heterozygote Differentiation
Example: Sequence variations (SNP GT) in the LPLH3 gene 72 samples, 164 bp amplicon heterozygous (homo and heteroduplexes) Gene Scanning High-Resolution Melting Analysis In this example, the “perfect” result of a Gene Scanning experiment is depicted. In the target LPLH3, a SNP from G into T results in such a destabilization of the amplicon, that both homoduplexes (wildtype and mutant) can be discriminated as well as the heterozygous types. homozygous wildtype (homoduplexes) homozygous mutant (homoduplexes)

Heterozygote Amplification
Observed Combination of 4 Duplexes Two Homoduplexes Two Heteroduplexes This slide shows differences between homo and hetero duplexes, and also how mixed sample has characteristics of both types of ds amplicons

Unlabeled Probe Genotyping and Amplicon Melting Simultaneous genotyping and scanning
Temperature Time (sec) 30 60 90 120 Scan the full fragment Genotype by probe melting People are starting to use unlabelled oligos to examine melt of oligo in combination amplicon melting to provide large amounts of information by a single reaction

Unlabeled Probe Melting Principle of Genotyping by Hi Res Melt
High-Resolution Melting with intercalating dye and unmodified oligo specific for known mutation site Same basic process as amplicon, except run reaction asymetically, that is, try to produce more of target strand where probe will bind

Combined Unlabeled Probe and Amplicon Melting Example 1: TNF
Probe for SNP CT Amplicon 136 bp 96 samples Wildtype 1st Derivative Mutation Heterozygotes Normalization, Difference Plot Homozygotes (not separated) Heterozygote Heterozygotes for another SNP (AG) in this amplicon Here is what these can look like This portion of first melt distinguishes between the homozygotes, and heterozygotes. The difference plot groups the homozygotes, but provides evidence of a different, unknown heterozygote (in the pink)

High Resolution Melting Utility
Optimization requirements Data and Results Possibilities References and Papers Utility –

High Resolution Melting Utility
Optimization requirements Data and Results Possibilities References and Papers Similar to qPCR, except that you should work towards most specific reaction, not most efficient

Optimizing a Gene Scanning Experiment MgCl2 Concentration
MWM PCR Products (+ NTC 4.0 mM) 50 bp 4.0 3.5 3.0 2.5 2.0 1.5 1.0 MgCl2 Concentration Agarose Gel 2% Determining the optimal MgCl2 concentration is essential to ensure both the specificity and robustness of the PCR. The optimum MgCl2 concentration for a LightCycler® 480 High Resolution Melting assay may vary from 1.5 to 3.5 mM. Therefore, we strongly recommend that you titrate the MgCl2 concentration in the reaction between 1.5 and 4.0 mM (in 0.5 mM steps) when establishing a new assay. Determine the specificity of each PCR by agarose gel electrophoresis or melt curve analysis. Difference of 12 cycles, 212 = 4100 times as much DNA produced 167 bp PCR Fragment MgCl2 Titration 1.0 – 4.0 mM PCR Primers: 200 nM each Touchdown PCR Protocol (64 – 54°C)

Optimizing a Gene Scanning Experiment Sample Material
Use consistent extraction protocols for all samples to be analyzed via High Resolution Melting. Quantify DNA samples using spectrophotometry. Adjust them to the same concentration prior to PCR Use the same amount of template in each reaction (5 to 30 ng template DNA in a 20 µl reaction). Amplification plots should produce a crossing point value of < 30. Crossing points (aka CT) should be within 5 cycles of each other Because a Gene Scanning experiment involves comparing melting profiles from independent PCR reactions, it is crucial to minimize reaction-to-reaction variability. Use the same extraction prodedure to prepare all samples to be analyzed via High Resolution Melting. For reproducible isolation of nucleic acids use: either the MagNA Pure LC Instrument or the MagNA Pure Compact Instrument together with a dedicated nucleic acid isolation kit (for automated isolation) or a HIGH PURE nucleic acid isolation kit (for manual isolation), e.g., the High Pure PCR Template Preparation Kit. Quantify DNA samples using spectrophotometry. Adjust them to the same concentration with the resuspension buffer. Salts affect DNA melting behavior, so it is important that the concentrations of buffer, Mg2+ and other salts in the reaction mix are as uniform as possible for all samples. Use the same amount of template in each reaction (5 to 30 ng template DNA in a 20 µl reaction). Amplification plots should produce a crossing point value of < 30.

Optimizinging a Gene Scanning Experiment PCR Primers
Design PCR primers that have annealing temperatures around 60°C and produce short amplicons, ideally100–250 bp. Use a software package to design primers Primer3 ( LightCycler® Probe Design Software 2.0. BLAST ( the primer sequences to ensure they are specific for the target species and gene. Use primers that have been purified by HPLC. Use low primer concentrations (e.g., 200 nM each) to avoid primer-dimer formation. Design PCR primers that have annealing temperatures around 60°C and produce short amplicons (100–250 bp). A single base variation affects the melting behavior of a 100 bp amplicon more than a 500 bp amplicon; thus, short amplicons are more likely to show the effects of small sequence changes. Nevertheless, it is possible to target longer sequences (up to 500 bp), but these products will usually have lower resolution. Use a software package like Primer3 ( or LightCycler® Probe Design Software 2.0 for designing the primers. BLAST ( the primer sequences to ensure they are specific for the target species and gene. Use primers that have been purified by HPLC. To avoid primer-dimer formation, use low primer concentrations (e.g., 200 nM each)

Optimizing a Gene Scanning Experiment PCR Programs: Amplification
Example: Touchdown PCR Touchdown PCR enables very specific reaction

Optimizing a Gene Scanning Experiment PCR Programs: High Resolution Melting
Example: HRM program Shorter melting curve (maybe); many more data points than SYBR melt

Optimizing a Gene-Scanning Experiment Controls
Negative Controls – ensure PCR products not result of carryover Positive Controls – may be eliminated if known reference standards are used Known Reference Genotypes – „Melt Standards“ Especially useful when only a few samples are compared or when unlabeled probes are used and designed against a specific sequence variant Replicates? Biological replicates can be used to provide an estimate of variation within a genotype Replicates of individual samples not required „experimental“ replicates used to confirm extraction / pipetting / PCR repeatability Controls:

Guidelines for successful HRM Assays
Analyze small DNA fragments There will be a bigger effect of a single base variation on a small amplicon. 2. Analyze a single pure product Primer-dimers and non-specific products make HRM difficult to interpret. 3. Use sufficient pre-amplification template Make sure the product has a Cp (CT) no more than 30 cycles. Samples that amplify later than this produce variable HRM results due to amplification artifacts. 4. Check for aberrant amplification plots Check the qPCR plots carefully for log-linear plots that are not steep, jagged, or reach a low signal plateau. This can indicate poor amplification, incorrect reaction setup, etc.

Guidelines for successful HRM Assays
5. Keep post-amplification sample concentrations similar The concentration of a DNA fragment affects its TM. Try to keep DNA concentrations as similar as possible. Make sure every reaction reached a plateau. 6. Ensure sample-to-sample uniformity All samples must be of equal volume and should contain the same concentration of dye. DNA melting behavior is affected by salts in the reaction mix so make sure the buffer, Mg and other salts is the same in all samples. Use identical tubes or plates for all comparisons. 7. Allow sufficient data collection for pre-and post-melt phases Collect HRM data points over about a 10o C window centered on the observed TM.

Optimizing a Gene-Scanning Experiment Troubleshooting – Montgomery et al (2007)
TABLE 1 | Troubleshooting table. Problem Possible reasons Solution Extraneous melting transitions or poor curve clustering Secondary PCR products Optimize PCR conditions to obtain clean product Low PCR yield Inconsistent genomic DNA preparation Optimize PCR to enhance product yield Ensure that the genomic DNA concentration and buffer is consistent Amplicon and probe melting transitions not visible or are very small PCR product Tm too high Probe Tm too high, preventing PCR extension Amplicon too long High GC content Redesign probe with lower Tm, use and exonuclease-positive Taq or add the probe after PCR Design primers for shorter amplicon length Add DMSO, betaine or glycerol to the PCR buffer

Optimizing a Gene-Scanning Experiment LightCycler® 480 High Resolution Master
Cat. No Kit for 5 x 100 reactions (20µL) Contents: Master Mix 2 x conc. contains FastStart Taq DNA Polymerase, reaction buffer, dNTP mix (with dUTP instead of dTTP), and ResoLight MgCl2, 25 mM to adjust MgCl2 concentration H2O, PCR-grade to adjust the final reaction volume Application For amplification and detection of a specific DNA sequence (with suitable primers) followed by high resolution melting curve analysis for detection of sequence variants among several samples. Our kit

Utility Optimization requirements Data and Results Possibilities
References and Papers Data from a customer in Atlanta GA

HRM 2 - Sensitivity testing Dilution series of wild type/mutant mixes
Another example of data – dilution series of wild type / mutant

HRM 3 - Sensitivity testing Mutations identified in 650bp product (samples shown in replicates)
606 G>A 434 C>G 421 C>T, 606 C>G Wild type Multiple SNPs in single product of note, two different C>T SNPs in slightly different areas of the amplicon, produce different melt curves

HRM of grape varieties Grape differences www.roche-applied-science.com
From: Plant Methods. 2008; 4: 8.

HRM Data – 5 DNA Methylation
The most basic analysis is done via the Gene Scanning module, which grouped similarly shaped melting profile together. Thus by comparing an unknown sample to a set of known standards one can get a semi-quantitative readout of the percentage methylation of the input DNA. Using this method as little as 0.5% methylated DNA produced a noticeably different melting profile for that of the pure unmethylated DNA. A little as 0.5% methylated DNA was detected UCLA

HRM Data – 6 Mycoplasma synoviae strain identification – Jeffery et al (2007)
Mycoplasma strain differentiation

References and Papers

High Resolution Melting Key applications
Scan genes to discover SNPs and/or somatic mutations Genotyping of known SNPs Characterization of haplotype blocks – “hap maps” DNA methylation analysis DNA mapping Species identification/taxonomy HLA compatibility Screening for loss of heterozygosity Association (case/control) studies Allelic prevalence in a population Identification of candidate predisposition genes Hap maps, strain id, hla compatibility, In a year, this list will be much bigger, and much more in depth

References and Papers These are the papers cited here; not only ones

HRM References Gundry CN, Vandersteen JG, Reed GH, Pryor RJ, Chen J, Wittwer CT. Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. Clin Chem Mar;49(3): Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem Jun;49(6 Pt 1): Zhou L, Myers AN, Vandersteen JG, Wang L, Wittwer CT. Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye. Clin Chem Aug;50(8): Zhou L, Wang L, Palais R, Pryor R, Wittwer CT. High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution. Clin Chem Oct;51(10): Jeffery N, Gasser R, Steer P, Noormohammadi A. Classification of Mycoplasma synoviae strains using single-strand conformation plolymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region. Microbiology ,

HRM References Fortini D, Ciammaruconi A, De Santis R, Fasanella A, Battisti A, D'Amelio R, Lista F, Cassone A, Carattoli A. Optimization of high-resolution melting analysis for low-cost and rapid screening of allelic variants of Bacillus anthracis by multiple-locus variable-number tandem repeat analysis. Clin Chem Jul;53(7): Vandersteen JG, Bayrak-Toydemir P, Palais RA, Wittwer CT. Identifying common genetic variants by high-resolution melting. Clin Chem Jul;53(7): Dobrowolski SF, Ellingson C, Coyne T, Grey J, Martin R, Naylor EW, Koch R, Levy HL. Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles. Mol Genet Metab Jul;91(3): Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 2007;35(6):e41. Montgomery J, Wittwer CT, Palais R, Zhou L. Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis. Nat Protoc. 2007;2(1):59-66.

HRM References von Ahsen, N. Two for typing: homogeneous combined single-nucleotide polymorphism scanning and genotyping. Clin Chem , Herrmann, M.G., Durtschi, J.D., Bromley, L.K., Wittwer, C.T. & Voelkerding, K.V. Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. Clin Chem , Dujols V, Kusukawa N, McKinney JT, Dobrowolsky SF, Wittwer CT. High-resolution melting analysis for scanning and genotyping., in Real Time PCR. Tevfik D, ed., Taylor and Francis, Abingdon, 2006. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004;50: Reischl U. Melting of the ribosomal RNA gene reveals bacterial species identity: a step toward a new rapid test in clinical microbiology. Clin Chem (11):

Why High Resolution Melting? Robust, non-destructive closed-tube method with many applications; highly informative and flexible. More convenient and cost-effective than current technologies, such as sequencing or dHPLC. Why HRM on the LightCycler® 480 System? Only plate-based Real-Time PCR HRM platform offering high-throughput HRM as a highly versatile, integrated system (hardware, software, reagents).

References and Papers What this means for other real time Applications Didn’t design 480 for HRM, but characteristics that make the 480 so accurate for qPCR make it possible to do HRM

Real Time PCR, HRM, and Quantification
The technological and biochemical requirements for accurate and meaningful HRM studies are fulfilled by the LC 480 system. HRM Scanning is another software module that expands the capabilities of the LC 480, the premier real time PCR system on the market. The technologies that enable HRM also provide unsurpassed accuracy and consistency for the amplification, producing excellent quantitative data and results.

The LightCycler® 480 System Data Homogeneity
“A Walk Around the Block” Standard curves

96-fold replicates of 3 genotypes
LightCycler® 480 Instrument Temperature Homogeneity 96-fold replicates of 3 genotypes Tm(1) / °C Tm(2) / °C average 56.47 64.88 minimum 56.14 64.67 maximum 56.85 65.4 delta 0.71 0.73 SD 0.1612 0.1801 Melt curves – look at SD Prototype Software

Thermal Homogeneity Demonstration by Melting Curve Analysis
   Tm-low Tm-med Tm-high Test to demonstrate thermal accuracy: SimpleProbe probes, Fluorescein label, 96 wells each, chequerboard pattern, 40°C to 90°C, 10 acquisitions/°C. The melting point temperature (Tm) of a given labeled oligonucleotide is used as a sensitive detection parameter (much more sensitive to variations than Cp values!) to demonstrate the temperature homogeneity across a multiwell plate. To cover different target temperatures, the experiment can be done with three different oligo/probe pairs …….. L/L T(52) M/M T(66) H/H T(78) SimpleProbe probes, FAM-label

Thermal Homogeneity - Experimental Setup Analysis of four 96-well Plate Subsets
A1: 96 x Tm-low (52°C) A2: 96 x Tm-high (78°C) …..added following a checkerboard pattern and run together on the same plate. Total samples B1: 96 x Tm-med (66°C) B2: 96 x negative control

Thermal Homogeneity – LightCycler 480 Intra-Run Reproducibility of 96 Replicates
Positions: A1-O23 Positions: B1-P23 Positions: A2-O24 These are the results for three different target temperatures on the LightCycler® 480 Instrument.

Data Uniformity Dilution Series/Neighboring Wells – 165 bp target
Experiment: Serial 10-fold dilutions 3 replicates Target: Cyp2C9.2; 165 bp long fragment Fast & Standard protocol (Hydrolysis Probe Format) Samples in neighboring wells Standard curve LightCycler® 480 (96): 55 min

Data Uniformity Two Copy Numbers/Spread Across Plate – 442 bp target
Experiment: Samples in checkerboard pattern 1000 & 100 copies 48 replicates Target: CycA; 442 bp long fragment Fast & Standard protocol (Hydrolysis Probe Format) Samples in neighboring wells LC 480 data LightCycler® 480 (96): 55 min

Data Uniformity Two Copy Numbers/Spread Across Plate – 442 bp target
ABI 7900 (96): 90 min AB 7900 (96) Fast: 44 min Experiment: Samples in checkerboard pattern 1000 & 100 copies 48 replicates Target: CycA; 442 bp long fragment Fast & Standard protocol (Hydrolysis Probe Format) Samples in neighboring wells AB data – same experiment

LightCycler® 480 System Applications
Gene Detection: Detecting e.g., bacteria in sample material Gene Expression: Analyzing expression level of gene of interest Genotyping: Detecting known variants Gene Scanning: Finding new variants

Credits Special thanks for contributions for this presentation:
Natalie Barnes – RAS Australia Systems Account Representative Dr. Michael Hoffman – RAS Global Marketing Manager Roche Applied Science US Technical Support Bill Demyan, Ph.D Joe Donnenhofer Alex Pierson Michelle Moore Duane Marks Dr. Oliver Geulen – RAS Global Training and Applications Steve Hurwitz – RAS US LightCycler Manager John Ogden, Ph.D – RAS US Genomics Marketing Manager People who helped with this presentation

HRM Genotyping – History, Technology, and Utility
Questions? Please feel free to contact us about any of our products: Field Applications Consultant x (voic ) Systems Account Manager x (voic )

LIGHTCYCLER, LC, HybProbe and SimpleProbe are trademarks of Roche

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