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If the OCP is complementary to the SNP, it can be circularized by DNA ligase (Fig.

An electrophoretic mobility shift for circularized OCP allowed quantification of ligation kinetics.

Analysis of the ERCA signal amplification step of the assay In reactions where the OCP matches the SNP, an estimated 100-30,000 OCPs are circularized by ligation depending on the amount of input DNA target.

In order to investigate the efficiency of the ERCA step, OCP was circularized by ligation on a synthetic oligonucleotide target, and gel-purified to remove any linear DNA.

The OCP ligation/ERCA method is based on ligation dependent circularization of allele specific DNA probes followed by exponential rolling circle amplification of the circularized probes [ 4 5 ] .

If the OCP and the target matched at the mutation site, the OCP was circularized by ligase.

Exponential amplification of circularized OCP requires primers P1 and P2 (see Figure 2).

Q-PNA Exponential amplification of circularized OCP (see Figure 2) was accomplished using generic primers P1 and P2, along with allele specific primer(s).

The reaction was heated to 95°C for 10 seconds, and cooled to 63-68°C for 5-20 minutes, during which time OCP annealed to genomic target and was circularized by ligase.

For each time point, the number of counts in unreacted intron, spliced intron, and circularized intron were determined.


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