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Application Note A08-004A Discrimination of a single base pair mismatch using

melting analysis

Introduction Melting analysis is frequently used to characterise PCR products post-amplification.

The melting temperature (Tm) of a PCR product is dependent on both the length and

sequence of the DNA and can therefore be used to check for non-specific products of a

reaction or to analyse DNA sequence variations such as insertions, deletions and single

base pair changes. Melting analysis requires the use of an intercalating dye such as

SYBR® Green I, SYTO® 9 or EvaGreen®. Intercalating dyes bind to the minor groove of

double-stranded DNA (dsDNA) producing up to a thousand-fold increase in

fluorescence. Third generation fluorescent dsDNA dyes such as SYTO® 9 and

EvaGreen® are able to be used at higher concentration in the reaction due to their

lower toxicity. This allows for greater saturation of the dsDNA leading to increased

sensitivity, high fidelity and better resolution in melting curve profiles.

In this application note we demonstrate that using melting analysis, PrimeQ was able to discriminate a single base

pair change (G/A conversion) using both SYBR® Green I and third generation intercalating dyes.

Methods Two artificial 100 base ssDNA templates were synthesized based on the genomic sequence of Enterobacteria

phage lambda (Figure 1). The lambda DNA sequence normally has a G base at position 16404. One of the templates

was synthesized with the G base in this position (“wild type”) and the second with an A in this position (“mutant”).

The primers were designed to amplify a 76 base pair product within the templates, encompassing the base change.

Selecting a short product ensures that the mismatch will have greatest effect on the overall Tm of the product.

Figure 1: Sequences of the lambda DNA templates and primers. The 100 base artificial templates were based on the sequence of bases 16361

to 16460 inclusive (accession number J02459.1). The “wild type” template had a G base at position 16404 and the “mutant” an A base.

Primers were designed using NCBI Primer-BLAST and are shown in blue and green.

Four different reagent kits were used for amplification and detection of the products (Table 1).

Kit name Supplier Part code DNA Polymerase Intercalating dye

SYBR® Green JumpStart™ Taq

ReadyMix™

Sigma S4438 JumpStart™ Taq SYBR® Green I

AccuMelt™ HRM SuperMix Quanta

Biosciences™

95103-250 AccuFast™ Taq DNA

Polymerase

SYTO® 9

Fast EvaGreen® qPCR Master Mix Biotium 31003 Cheetah™ Hot Start Taq DNA

Polymerase

EvaGreen®

QuantiFast® SYBR® Green PCR Kit Qiagen 204052 HotStarTaq® Plus DNA

Polymerase

SYBR® Green I

Table 1: Details of the reagent kits used. All kits were supplied as complete 2X master mixes.

Approximately 5 x 105 copies of either template were used per reaction. Six replicates of either template were run

for each kit plus two no template controls (NTC). The final primer concentration was 0.2µM and ROX™ passive

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A08-004A: Discrimination of a single base pair mismatch

reference dye (PRD) was added to each mix to a final concentration of 40nM except QuantiFast® (which already

contained a PRD). White, low profile 96-well plates were used (Brand; part code 781365) and heat sealed with

optically clear heat sealing film (Clear Seal Diamond, Thermo Scientific; part code AB-0812). The reactions were

amplified for 35 cycles (initial denaturation, 95°C, 2 min; 35 cycles of 95°C, 5s, 60°C, 20s reading with filter sets

FC02 and FC04). At the end of the amplification stage, a melting analysis stage was performed (72°C to 91°C in

0.2°C steps, hold time 10 seconds per step, reading with filter set FC02).

Results Single base changes in DNA, sometimes known as SNPs (single nucleotide polymorphisms) are classified into 4

groups as summarised in Table 2.

SNP class Base change Typical Tm curve shift Frequency in the human

genome

1 C/T and G/A Large (>0.5°C) 64%

2 C/A and G/T Large (>0.5°C) 20%

3 C/G Small (0.2-0.5°C) 9%

4 A/T Small (<0.2°) 7%

Table 2: SNP classes and occurrence in the human genome (1).

G to A base changes are designated as class 1 SNPs and are one of the easiest types to detect due to the relatively

large shift in Tm; they are also one of the most frequently found class of SNPs in the human genome. Figure 2 shows

the amplification curves of the products obtained from all the kits on the same scale with no PRD correction. It is

quite clear that kits containing third generation dsDNA dyes (AccuMelt™ and Fast EvaGreen®) show a much greater

final fluorescence than those containing SYBR® Green I. The SYBR® Green containing kits reached a plateau at a

much earlier cycle number whereas the SYTO® 9 and EvaGreen® kits continued to amplify in an extended linear

phase before reaching a final plateau.

The melting stage was analysed using the dissociation curve analysis in Quansoft and Figure 3 shows the melting

curves and melting peaks for both the “wild type” and “mutant” templates using each of the kits. In each case, the

six replicates for each template type were clearly distinct. For further analysis, the raw fluorescence data for the

samples amplified using the QuantiFast® kit were exported to Excel. The melting curves were normalised between

78°C and 91°C such that the fluorescent signal at 78°C was set to 100% and the signal at 91°C, 0%. This helps to

“tidy” the data and aids in interpretation. To further enhance the differences between curves, they can be plotted

Amplification

Cycle Number34323028262422201816

Relative Fluorescence

700,000

650,000

600,000

550,000

500,000

450,000

400,000

350,000

300,000

250,000

200,000

150,000

100,000

50,000

0

Figure 2: Amplification curves for the two templates with each of the

kits. Red = AccuMelt™, purple = EvaGreen®, green = JumpStart™, yellow

= QuantiFast®, blue = NTCs. The NTCs showed a small increase in

amplification towards the end of the run due to non-specific

amplification.

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A08-004A: Discrimination of a single base pair mismatch

on a difference graph. A curve is selected as a reference and the difference in fluorescence between the samples

and reference is plotted. These two graphs are shown in Figure 4.

Figure 3: Melting curves and melting peaks for each of the templates obtained with the reagents used. In each case the “wild type”, G

template is shown in green and the “mutant”, A template in red. As expected, the “mutant” template has a lower Tm.

Figure 4: Normalised melting curve data and difference curves for “wild type” and “mutant” samples amplified using the QuantiFast® SYBR®

Green kit. The reference used for the difference plots was one of the “wild type” melting curves. These two plots clearly demonstrate the

difference in Tm between the two sets of samples.

The Tm of a product is strongly influenced by the reaction mixes, which may vary in unspecified additives and

differences in salt concentrations and this is apparent from the data presented in Figure 5 and Table 3, where the

Tm for the “wild type” sample was found to vary between 81.61°C and 83.76°C. The shift in Tm (ΔTm) also varied

quite significantly depending on which kit was used. Of the reagents tested, those containing SYBR® Green I

showed the greatest shift in Tm between the “wild type” and “mutant” PCR products in this particular assay.

JumpStart™ AccuMelt™ Fast EvaGreen® QuantiFast®

G A G A G A G A

Average Tm

+/-SD (°C)

83.20 +/-

0.019

82.43 +/-

0.050

83.76 +/-

0.094

83.21 +/-

0.040

81.61 +/-

0.031

80.87 +/-

0.082

82.90 +/-

0.021

82.03 +/-

0.061

ΔTm (°C) 0.77 0.55 0.74 0.87

Dye SYBR® Green I SYTO® 9 EvaGreen® SYBR® Green I

Table 3: Data from Figure 5 showing the Tm shift observed with each of the kits for the single base mismatch.

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A08-004A: Discrimination of a single base pair mismatch

Conclusions Melting analysis in qPCR has traditionally been used to identify non-specific products or contamination following

amplification. A melting curve is generated by slowly denaturing the DNA in the presence of a dsDNA binding dye.

Small variations in sequence result in differences in Tm, which is defined as the point at which 50% of the DNA is

double stranded and 50% is single stranded. High Resolution Melting (HRM) is an emerging technique which uses

these small changes in Tm to characterize genetic variations. Using this technique, fluorescence data is collected at

very small temperature increments in order to obtain the highest resolution melting curves. Special software is also

required in order to analyse fully the subtle differences in curve shape or Tm differences of products. This,

combined with reagents and dyes designed specifically for HRM, results in a powerful technique for high

throughput screening for genetic variation.

In this application note we designed a class 1 SNP variation and demonstrated that PrimeQ is able to distinguish a

single base pair mismatch (G/A) using the standard melting curve analysis software available in Quansoft. This

analysis can be further enhanced by exporting the raw data to Microsoft® Excel in order to generate normalised

and difference curves. In addition, the products were easily distinguishable using either SYBR® Green I or the third

generation intercalating dyes SYTO® 9 and EvaGreen®. Class 1 and 2 SNPs are the easiest to detect due to the

relatively large Tm shift; we have not tested class 3 or 4 SNPs as these are relatively rare and the typical ΔTm

(~0.2°C) might prove difficult to interpret due to being right at the edge of the capability of the thermal block of

PrimeQ.

References (1) J. Craig Venter et al. The Sequence of the Human Genome Science 291, 1304 (2001);

DOI:10.1126/science.1058040

Trademarks

EvaGreen® is a registered trademark of Biotium, Inc.

SYBR® is a registered trademark of Molecular Probes, Inc.

ROX™ is a trademark of Applera Corporation.

QuantiFast® is a registered trademark of Qiagen Group

AccuMelt™ is a trademark of Quanta BioSciences, Inc.

SYTO® is a registered trademark of Life Technologies Corporation (Molecular Probes Labelling and Detection Technologies).

JumpStart™ is a trademark of Sigma-Aldrich Co. LLC

80.00

80.50

81.00

81.50

82.00

82.50

83.00

83.50

84.00

84.50

G A G A G A G A

JumpStart™ AccuMelt™ EvaGreen® QuantiFast®

Tm

(°C

)

Figure 5: Tm values for the PCR products determined in each of

the reaction mixes.