Melting curves probes

One way to obtain discrimination between mismatched and complementary strands is the use of high temperatures during the hybridisation reaction. The difference in the melting curves of the target from perfectly matched and mismatched capture probes can allow to define a specific temperature to discriminate perfect matches and mismatches. However, the employment of high temperatures during hybridisation step requires precise temperature control, which is difficult to achieve and is expensive [48]. 

Figure 3. Melting curves for different surface probe densities (coverage) as indicated. In solution Tm = AH0/(AS0 - RlnC), W = ARTfJAHo. ...

Figure 37-19 Melting curve of double-helical nucleic acid. (Modified with permission from Piper MA, Unger ER Nucleic Add Probes A Primer for Potho/og/sts. Chicago, ASCP Press, 1989.)...

Figure 37-29 Melting curve 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. (Modified with perm/ss/on of the publisher from Bernard PS, Pritham GH, Wittwer CT Color multiplexing fiybr/d/zat/on probes using the opo/ipoprotein /ocus as a model system for genotyping. Anal Biochem 1999,2 73 221-228. 1999 Academic Press.)...

Bullock GC> Bruns DE, Haverstick DM, Hepatitis C genotype determination by melting curve analysis with a single set of fluorescence resonance energy transfer probes. Clin Chem 2002 48 2147-54. 

Fig. 1. Derivative melting curves using ITI simple probes, showing wild-type (wt) and variant alleles. Heterozygous, no template control. In each case, the probe is a perfect match to the variant allele. Heterozygous (black line), no template control (grey line).

Rowe LR, Bentz BG, Bentz JS. Detection of BRAF V600E activating mutation in papillary thyroid carcinoma using PCR with allele-specific fluorescent probe melting curve analysis. J Clin Pathol. 2007 60 1211-1215. 

Figure 7. Melting curves for matched probes obtained at target concentrations of (a) 10 nM, (b) 150 nM, and (c) 500 nM.

Figure 8. Melting curves obtained in high-low and low-high temperature directions on the same set of matched probe synthesis sites at (a) 10 nM and (b) 500 nM.

Figure 9. Melting curves for matched and single-base mismatched probes (position 10) obtained at a target concentration of 50 nM illustrated for (a) 10-mer and (b) 20-mer probes.

Some of the broadening and depression of the melting curves has been shown to be a consequence of the presence of truncated probes. The melting behavior of probes synthesised by alternate methodoligies with higher stepwise yields should permit the... 

Figure 13. Melting curves expected for synthesis sites with the probe length distributions of Figure 12 assuming solution phase energetics, Langmuir adsorption, and 10 nM target concentration.

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