Extension PCR

Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P., and Johnson, J. M. (2005). Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737-1744. 

Fig. 9.1 (continued) antibody domain in the second overlap-extension PCR. This second PCR yields cloning-ready anti body domains with flanking restriction sites and combinatoriaUy shuffled CDRs. This scheme is for VH domains and identical scheme with the appropriate primer sets was used to prepare the light chain domains. RE 5 represents a restriction site for Ncol for VH domains or EcoRW for VL domains. RE 3 represents a restriction site for Blpl for VH domains or Notl for VL domains... 

N, where N is the number of cycles. The choice of temperatures and timing is critical as is the choice of primers and their concentration. For example, if sufficient time is not allowed for replication, then partially completed DNA fragments will be present and could act as primers in subsequent cycles — this phenomenon is used to advantage in the staggered extension PCR (StEP) technique to be described later. 

Figure 6 A schematic diagram showing how recombination is achieved using staggered extension PCR (StEP).

Fig. 3. Mutagenesis by overlap extension PCR [22]. PCR products are shown as two paired strands primers are shown as horizontal arrows and mutations in primers and products as black dots...

Key words Overlap extension PCR, GC-rich annealing sequences. Recombinant DNA construction. 

FIGURE A.6 An alternative overlap-extension PCR mutagenesis scheme. The method employs a single mutagenic primer (mj) and three universal primers (U], U2, and U3). Note that the 5 end of primer uj contains mismatches (8 nt) designed to prevent the termini extension in the second PCR. 

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