FIGURE 10-6 Adding EcoRI sites to the ends of PCR products. (a) A pair of PCR primers is designed so that their 3' ends anneal to the target sequence while their 5' ends contain sequances encoding the restriction enzyme site (EcoRI in this case). Two additional (random) nucleotides are added to the 5' end because restriction enzymes require sequences on both sides of the recognition sequence for efficient cutting. The target DNA is denatured, and 5' ends with the restriction sites remain single stranded while the rest of the primers anneal and are extended by DNA polymerase. (b) In the second round of PCR-only the newly synthesized strands are shown-the DNA primers anneal again, and this time DNA synthesis produces double-stranded DNA molecules just like conventional PCR, but Producing PCR products with sticky ends Initial steps of PCR (heat to 95°C, then cool to anneal and synthesize DNA) (a) CITAAGCG 5 5 GCGAATTC these molecules have restriction sites at one end. (c) The products of the second round and all subsequent rounds have EcoRI sites at both ends. (d) When these are cut with EcoRI, sticky ends are produced. Second round of PCR (b) GCGAATTC CTTAAGCE 5' 5 GCGAATTC CTTAAGCG endonuclease recognition sequences at their 5' ends (Figure 10-6). Digestion of the final PCR product with the restriction enzyme (EcoRI in this case) produces a fragment that is ready to be inserted into a vector (see Figure 10-5b). Another method adds sticky ends to any double-stranded DNA fragment-including cDNAs (Figure 10-7). Short double-stranded oligonucleotides (called linkers or adapters) that contain a restric- tion site are added to a test tube containing cDNAs and ligase. The ligase joins the linkers to the ends of the CDNA strands. After liga- tion is complete, the DNA is incubated with the corresponding restriction enzyme to generate the sticky ends necessary for clon- ing into a plasmid vector (see Figure 10-5b). Note that in the exam- ples shown, both the amplified DNA and the CDNA must not contain an internal EcoRI site or it too will be digested. If it does, Further rounds of PCR (c) GCGAATTC GAATTCGC CTTAAGCG CGGTTAAG Digest with EcoRI (d) 5' AATTC CITAA 5'