Principles of scanning by melting

The principle behind heterozygote detection by melting is shown in Figure 9.2. PCR amplification of a heterozygous SNP produces four different single-stranded products, indicated as A, C, G, and T in Figure 9.2. If these strands are randomly associated after PCR by denaturation and annealing, four unique duplexes are formed. Two fully base paired fragments are identical to the starting DNA and are close (but usually not identical) in melting temperature. In addition, two imperfectly matched heteroduplexes are

Observed combination of four duplexes mil

Observed combination of four duplexes mil

Temperature

Figure 9.2

Heterozygote detection by melting analysis. When a heterozygous sample is amplified by PCR, denatured, and cooled, the strands re-associate randomly, producing four distinct duplexes. The two homoduplexes and two heteroduplexes that are produced cannot be observed individually during melting. Rather, the composite melting curve of all four duplexes results in a skewed melting curve with a broad melting transition. The altered shape of the melting curve is used to identify heterozygotes.

formed that melt at lower temperatures than the fully base paired fragments. All four duplexes are present when a heterozygous sample is melted, so the observed melting curve is a superposition of all four melting profiles. The result is usually a skewed melting curve with a broad transition. The best indicator for the presence of a heterozygous sample is the altered shape of the melting profile. The melting temperature, or Tm, is less sensitive and not as relevant as melting curve shape.

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