Figure 3

Illustration of peak resolution in CTCE. A. Separation of homoduplexes and heteroduplexes as electophoretic peaks using target sequence #1 from the gene BRCA1. The melting profile was irregular with a mean melting temperature of 70.7°C for the wild type homoduplex (peak 1). Twenty one-minute temperature cycles between 47°C and 59°C yielded baseline separations between homoduplexes and heteroduplexes. Small diffuse peaks result from errors generated during PCR with low-fidelity Taq polymerase and chemical reactions, such as thermal deamination of cytosine. These are avoided in mutation detection protocols that minimize the effect of deamination and employ Pfu DNA polymerase which does not copy passed a deaminated cytosine (uracil). B. Effect of mean column temperature on peak resolution. Temperature was varied in twenty one-minute cycles with 3°C amplitude and mean temperatures varying from 41.5 to 57.5°C.