Relative quantification of different gene modifications amplification deletion rearrangement translocation

The choice of the most appropriate conditions for these types of assay corresponds to the combination of primer concentration allowing a ratio of 1 between the target gene and the reference gene in a DNA sample with no genetic abnormality. This ratio is calculated using the ACt method (ACt = Ct of target - Ct of reference = 0), a ACt of 0 indicating a ratio of 1. A number of factors can influence this assay, and the optimization consists of compensating these factors to obtain a ratio of 1 in a DNA sample with no genetic abnormality. The inclusion of normal DNA sample in each experiment also provides a reference sample for a AACt method of calculation. As the normalization will relate to a value close to 0 for the second ACt, both methods essentially give the same result. For an accurate quantification it is also necessary to verify that PCR efficiency is independent of the initial amount of target DNA. Each pair of primers therefore needs to be tested across several log dilution of a control DNA sample as described above.

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Primer-dimer

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78.5

87.5

1 1 1 1 1 1 1 1

| i i i i | i i i i | i i i i | i i i i | i i

65 70 75 80 85 90 95

Melting temperature (°C)

65 70 75 80 85 90 95

Melting temperature (°C)

Figure 8.5

An illustration of the use of melting curve analysis to distinguish between specific and non-specific PCR products (see text).

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