Ligate amplicon

Amplification is achieved by repeated cycles (20 to 50) of heating (for strand separation) and cooling (for specific hybridization). Amplicons result upon ligation of adjacent probes. A commercialized automated system achieves detection... 

Fig. 1. Adaptation of anti-cell aptamers to PLA. Anti-PSMA aptamers were extended at the 3 and the 5 end and DNA probes were hybridized to the extended aptamers. When bound to adjacent sites on a cell surface the oligonucleotides (one of which contained a 5 phosphate) could be aligned by a splint oligonucleotide and subsequently be ligated and amplified. The new amplicon is detected by real-time PCR.

The second area is a PCR Staging Room in which the products from the ligation stage are prepped ready for the PCR reaction. The area should be essentially amplicon-free. It is acceptable to have the pre-PCR Clean Room and PCR-Staging room as one area, providing the workflow is unidirectional. This room (or area) should also contain any necessary reagents to set up the PCR but thermo-cycling amplification steps MUST be undertaken elsewhere (e.g., in the Main Lab ). 

Ligate the target locus with T4 DNA ligase to the restricted and cleaned pOK12 amplicon overnight at 16°C. 

Ligate the amplicons to digested resistance cassettes with T4 DNA ligase at 16°C overnight, and transform into TOPIO E. coli as described in step 10, Subheading 3.1.1. 

The volnme of PCR reactions used for the ligation can range from 0.5 pi to 4 pi depending on the yield of the amplicons. 

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