Gene cloning is used to make many copies of a gene and to produce a protein product from it. The gene that makes a desired protein can be transferred to a new species or individual using a recombinant DNA plasmid. To isolate a gene for cloning, genetic engineers use restriction enzymes to cut DNA strands at specific DNA sequences called restriction sites. The process of making a recombinant DNA plasmid using restriction enzymes is depicted in figure 1: A bacterial plasmid shows a restriction site of GAATTC with complementary DNA sequence of CTTAAG. In step 1, a restriction enzyme cuts the sugar phosphate backbones at each arrow, which creates sticky ends TTAA and AATT. In step 2, a DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. A fragment from a different DNA molecule cut by the same restriction enzyme has sticky ends AATT and TTAA. One possible combination matches the sticky ends of the new target DNA onto the plasmid DNA. In step 3, DNA ligase seals the strands together, forming a new recombinant plasmid with the new target DNA inside.

Figure 1: The restriction enzyme in this example (called EcoRI) recognizes a single six-base-pair restriction site present in the plasmid. It makes staggered cuts in the sugar-phosphate backbones, producing fragments with "sticky ends." Foreign DNA fragments with complementary sticky ends can base-pair with the plasmid ends; the ligated product is a recombinant plasmid.

Describe the biological process that clones the recombinant plasmid in the model, and identify the eukaryotic cell location of the biological processes.
Explain the relationship between the restriction sites and the base pairing of sticky ends in the model. Explain the importance of using the same restriction enzyme for the target DNA and the plasmid DNA.
The sticky end of a target DNA sequence is ATTG. Identify the complementary sticky end of the plasmid DNA sequence. Predict what would happen if a mutation occurs in this DNA sequence.
Justify your prediction