Lakhasly

Melting curve analysis

Melting temperature o Each double strand DNA (ds DNA) has its own specific melting temperature (Tm), which is defined as the temperature at which 50% of the DNA becomes single stranded.o The temperature at which 50% of strands are hybridized is Tm (melting temperature), which is specific for each sequence, in this case is 81.5?C

Melting curve analysis (cont.) o After mathematical processing of such data (arising from fluorescence changes vs. derivative of the temperature, dF/dT), we obtain the specific fluorescence data, Rn (left axis).Calculate Tm o We use the following modified Marmur Doty formula: o Tm = 2(A + T) + 4(C + G) - 7

o Where: Tm = melting temperature in ?C A = number of adenosine nucleotides in the sequence T = number of thymidine nucleotides in the sequence C = number of cytidine nucleotides in the sequence

G = number of guanosine nucleotides in the sequence -7 = correction factor accounting for in solution o Marmur Doty Example Calculation.This example will demonstrate the manual calculation of the Tm for the following sequence: o 5'-ACGTCCGGACTT-3' o Step one: plug values into Marmur Doty formula to calculate melting temperature

o Tm = 2(A + T) + 4(C + G) - 7 o Tm = 2(2 + 3) + 4(4 + 3) - 7 o Tm = 31.0 ?C

o Where: Tm = melting temperature in ?C ?H = enthalpy change in kcal mol-1 (accounts for the energy change during annealing / melting) A = constant of -0.0108 kcal K-1 ?mol-1 (constant that scales energy to temperature) C = oligonucleotide concentration in M or mol L-1 (we use 0.0000005, i.e. 0.5 uM) -273.15 = conversion factor to change the expected temperature in Kelvins to ?C

[Na+] = sodium ion concentration in M or mol L-1 (we use 0.05, i.e. 50 mM)

One peak = One amplicon, or does it?o Researchers often use melting curve analysis to determine the specificity of the qPCR assay, o One of the other advantage of using the melt curve analysis method is that to find the Single nucleotide Polymerphisms (SNPs).Melting protocols (cont.) o As the temperature increases, dsDNA denatures becoming singlestranded, and the dye dissociates, resulting in decreasing fluorescence (Figure).Melting protocols o Typically, the thermal cycler being used to run the qPCR is programmed to produce the melt curve after the amplification cycles are completed.SNP detection by melting curve analysis o Melting curve analysis exploits the fact that even a single mismatch between the labeled probe and the amplicon will significantly reduce the melting temperature.o Thus, probe/amplicon heteroduplexes containing mismatches, such as the SNP, melt off at lower temperatures than probes bound to a perfectly matched target DNA (i.e., wild type).o Immediately after the last PCR cycle, the samples are momentarily denatured (90 ?C to 94 ?C), the thermal cycler starts at a preset temperature (usually above the primer T m ; e.g., 65?C) and measures the amount of fluorescence.o Melt curve analysis method can be used to check for any primer dimer formation and other anomalies in the qPCR assay developed.Melting curve analysis o The dotted line shows the fluorescence during the heating process; at low temperatures DNA is in double strand form and it has a 100% fluorescence (right axis).o Thus, there are two peaks, the lower peak at left, 72?C, corresponding to the dissociation curve of primer dimers that could be formed during the reaction. degree of GC content (Tm is higher in GC-rich fragments due to the presence of 3 hydrogen bonds) o The reason for this is due to the presence of 3 hydrogen bonds in the GC Pairing as compared to two hydrogen bonds in the AT pairing.mol-1 (accounts for helix initiation during annealing / melting) ?S = entropy change in kcal K-1 ?o The peaks on the right at 81.5?C which show higher intensity, corresponding to the dissociation curve of two specific amplification products obtained.o The temperature of the sample is then increased incrementally as the instrument (temp rate of 0.1 ?C to 0.3 ?C/Second) while continues to measure fluorescence.o The change in slope of this curve is then plotted as a function of temperature to obtain the melt curve for CFTR exon 17b (Figure 1A).mol-1 (accounts for energy unable to do work, i.e. disorder) R = gas constant of 0.00199 kcal K-1 ?o A well-optimized probe design will provide a Tm difference of 8 ?C to -10 ?C for a single base mismatch under the probe.o These melting temperatures are primarily determined by ?o As they heat, the denatured strands produce fewer signal.dsDNA length, ?and degree of complementarity between strands.o So more energy is to break the GC pair.?

Melting curve analysis

Melting temperature
• Each double strand DNA (ds DNA) has its own specific melting
temperature (Tm), which is defined as the temperature at which 50%
of the DNA becomes single stranded.
• These melting temperatures are primarily determined by
❖ dsDNA length,
❖ degree of GC content (Tm is higher in GC-rich fragments due to the presence
of 3 hydrogen bonds)
• The reason for this is due to the presence of 3 hydrogen bonds in the GC
Pairing as compared to two hydrogen bonds in the AT pairing.
• So more energy is to break the GC pair.
❖ and degree of complementarity between strands.

Calculate Tm
• We use the following modified Marmur Doty formula:
• Tm = 2(A + T) + 4(C + G) - 7

• Where:
Tm = melting temperature in °C
A = number of adenosine nucleotides in the sequence
T = number of thymidine nucleotides in the sequence
C = number of cytidine nucleotides in the sequence

G = number of guanosine nucleotides in the sequence
-7 = correction factor accounting for in solution
• Marmur Doty Example Calculation. This example will demonstrate the manual calculation of the Tm for the following sequence:
• 5'-ACGTCCGGACTT-3'
• Step one: plug values into Marmur Doty formula to calculate melting temperature

• Tm = 2(A + T) + 4(C + G) - 7
• Tm = 2(2 + 3) + 4(4 + 3) - 7
• Tm = 31.0 °C

• Where:
Tm = melting temperature in °C
ΔH = enthalpy change in kcal mol-1 (accounts for the energy change during annealing / melting)
A = constant of -0.0108 kcal K-1 ᐧ mol-1 (accounts for helix initiation during annealing / melting)
ΔS = entropy change in kcal K-1 ᐧ mol-1 (accounts for energy unable to do work, i.e. disorder)
R = gas constant of 0.00199 kcal K-1 ᐧ mol-1 (constant that scales energy to temperature)
C = oligonucleotide concentration in M or mol L-1 (we use 0.0000005, i.e. 0.5 µM)
-273.15 = conversion factor to change the expected temperature in Kelvins to °C

[Na+] = sodium ion concentration in M or mol L-1 (we use 0.05, i.e. 50 mM)

One peak = One amplicon, or does it?
• Melt curve analysis method can be used to check for any primer dimer
formation and other anomalies in the qPCR assay developed.
• Researchers often use melting curve analysis to determine the specificity
of the qPCR assay,
• One of the other advantage of using the melt curve analysis method is
that to find the Single nucleotide Polymerphisms (SNPs).

Melting curve analysis
• The dotted line shows the fluorescence during the heating
process; at low temperatures DNA is in double strand form and
it has a 100% fluorescence (right axis).
• As they heat, the denatured strands produce fewer signal.
• The temperature at which 50% of strands are hybridized is Tm
(melting temperature), which is specific for each sequence, in
this case is 81.5°C

Melting curve analysis (cont.)
• After mathematical processing of such data
(arising from fluorescence changes vs. derivative of the
temperature, dF/dT), we obtain the specific fluorescence data, Rn
(left axis).
• Thus, there are two peaks, the lower peak at left, 72°C,
corresponding to the dissociation curve of primer dimers that
could be formed during the reaction.
• The peaks on the right at 81.5°C which show higher intensity,
corresponding to the dissociation curve of two specific
amplification products obtained.

Melting protocols
• Typically, the thermal cycler being used to run the qPCR is programmed to
produce the melt curve after the amplification cycles are completed.

• Immediately after the last PCR cycle, the samples are momentarily
denatured (90 °C to 94 °C), the thermal cycler starts at a preset
temperature (usually above the primer T m ; e.g., 65°C) and measures the
amount of fluorescence.
• The temperature of the sample is then increased incrementally as the
instrument (temp rate of 0.1 °C to 0.3 °C/Second) while continues to
measure fluorescence.

Melting protocols (cont.)
• As the temperature increases, dsDNA denatures becoming singlestranded, and the dye dissociates, resulting in decreasing
fluorescence (Figure).
• The change in slope of this curve is then plotted as a function of
temperature to obtain the melt curve for CFTR exon 17b (Figure 1A).

SNP detection by melting curve analysis
• Melting curve analysis exploits the fact that even a single mismatch between the
labeled probe and the amplicon will significantly reduce the melting temperature.
• Thus, probe/amplicon heteroduplexes containing mismatches, such as the SNP,
melt off at lower temperatures than probes bound to a perfectly matched target
DNA (i.e., wild type).
• A well-optimized probe design will provide a Tm difference of 8 °C to -10 °C for a
single base mismatch under the probe.
• This difference in Tm is detected in a "melting curve" that measures changes in
fluorescence as the samples are heated.
• By taking the negative first derivative (-dF/dT) of the melting curve, the melting
temperature of the hybrids can be easily visualized and compared, simplifying the
discrimination of wild-type, heterozygous, and homozygous mutant samples .

Melting curve single-nucleotide
polymorphism (SNP) genotyping. A
heterozygous specimen with an SNP
under the probe is amplified and
melted. Two temperature transitions
are visible: one from the mutant allele
that is mismatched with the probe and
melts at a lower temperature, and one
from the normal allele that is
completely matched with the probe and
melts at a higher temperature. The
derivative plot shows the melting
temperatures of both the mutant-probe
and the normal-probe duplexes as
peaks.

Single probe PCR melting curve analysis MTHFR C677T SNP sites
• Folic acid, is a water-soluble vitamin known for its abundance in green leaves.
• It is particularly important for pregnant women since it has crucial roles during
pregnancy and fetal development.
• Folate metabolic disorders can cause fetal open neural tube defects
(e.g., anencephaly, spina bifida), Down syndrome, congenital heart disease, and
birth defects such as cleft lip and palate, fetal developmental delays, prematurity,
and low birth weight, and in pregnant women, these can lead to recurrent
abortion, gestational hypertension, preeclampsia, and placenta dissection.
• The 5,10-methylenetetrahydrofolate reductase (MTHFR) locus is mapped
on chromosome 1 at the end of the short arm (1p36.6).
• MTHFR is an important enzyme involved in the metabolism of folic acid,
synthesis of DNA, and DNA methylation, and it mainly converts 5,10methylenetetrahydrofolate to 5-methyltetrahydrofolate, which has a biological
function.

Single probe PCR melting curve analysis
MTHFR C677T SNP
• Several studies have used PCR to detect the SNP sites of MTHFR C677T.
• The existing detection methods for MTHFR C677T are
• (1) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), chip
hybridization, ……
• (2) Fluorescence probe PCR method
• (3) Fluorescence PCR melting curve method.
• The melting curve method has been used to detect SNPs with only one fluorescent probe, which can
detect the wild-type and mutant SNP sites.
• The melting curve method is more useful in clinical practice than the
traditional TaqMan fluorescent probe method, and in addition, rare and unknown SNP sites may be
discovered.
• However, for SNP detection, purified nucleic acids must be used as templates, which increases the
detection time and cost.
• Therefore, for a quicker and more convenient detection, we used a single-probe PCR method
combined with the melting curve to accurately analyze the MTHFR C677T SNP sites.

Evaluating the effectiveness of the PCR melting
curve method
• After researching numerous previous studies, it was found that when the peak
value of the melting curve T (m) of the PCR product was less than 50, false
positive results might have occurred.
• Therefore, a prerequisite for our result interpretation is that the peak value of the
melting curve T (m) must be greater than 50.
• According to the design of the fluorescent probes, the peak T (m) of the melting curve of C677C
(wild type) was 53 ± 1.0 °C,
• the peak T (m) of the melting curve of C677T (mutant) was 61 ± 1.0 °C,
• and the double peak T (m) of the melting curve of the heterozygous type was 53 ± 1.0 °C and
61 ± 1.0 °C, respectively.
• PCR results showed that T (m) peaks, and dissolution curves of C677C, C677T, and heterozygous
type were consistent with the predicted values (Fig. 1A).
• Notably, the T (m) peak values of these melting curves exceeded 50.