Chapter Summary

• Recombinant DNA is formed in the laboratory by combining at least two DNA sequences from different sources. Review Figure 18.1

• Many restriction enzymes make staggered cuts in the two strands of DNA, creating fragments that have sticky ends with unpaired bases.

• DNA fragments cut with restriction enzymes can be used to create recombinant DNA. DNA molecules from different sources can be cut with the same restriction enzyme and spliced together using DNA ligase. Review Focus: Key Figure 18.2

• One reason to make recombinant DNA is to clone a particular gene or other DNA sequence, either for sequencing and subsequent analysis or to produce a protein product in quantity.

• Bacteria, yeasts, and cultured plant and animal cells are commonly used as hosts for recombinant DNA. The insertion of foreign DNA into host cells is called transformation or (for animal cells) transfection. Transformed or transfected cells are called transgenic cells.

• Various methods are used to get recombinant DNA into cells. These include chemical or electrical treatment of the cells, the use of viral vectors, and injection. Agrobacterium tumefaciens is often used to insert DNA into plant cells.

• To identify host cells that have taken up a foreign gene, the inserted sequence can be tagged with one or more reporter genes, which are genetic markers with easily identifiable phenotypes. Selectable markers allow for the selective growth of transgenic cells. Review Figures 18.3, 18.4

• Replication of the foreign gene in the host cell requires that it become part of a segment of DNA that contains a replicon (origin and terminus of replication).

• Vectors are DNA sequences that can carry new DNA into host cells. Plasmids and viruses are commonly used as vectors.

• DNA fragments from a genome can be inserted into host cells to create a genomic library. Review Figure 18.5A

• The mRNAs produced in a certain tissue at a certain time can be extracted and used to create complementary DNA (cDNA) by reverse transcription. Review Figure 18.5B

• PCR products can be used for cloning. Synthetic DNA containing any desired sequence can be made in the laboratory.

• CRISPR technology can be used to inactivate or mutated any gene. Review Figure 18.6, Activity 18.1

• Gene silencing techniques can be used to inactivate the mRNA transcript of a gene, which may provide clues to the gene’s function. Artificially created antisense RNA or siRNA can be added to a cell to prevent translation of a specific mRNA. Review Figure 18.7

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• DNA microarray technology permits the screening of thousands of cDNA sequences at the same time. Review Figure 18.8, Animation 18.2

• Biotechnology is the use of living cells to produce materials useful to people.

• Expression vectors allow a transgene to be expressed in a host cell. Review Figure 18.9, Activity 18.1

• Recombinant DNA techniques have been used to make medically useful proteins. Review Investigating Life: Producing TPA

• Pharming is the use of transgenic plants or animals to produce pharmaceuticals. Review Figure 18.10

• Because recombinant DNA technology has several advantages over traditional agricultural biotechnology, it is being extensively applied to agriculture.

• Biotechnology may allow us to adapt the plant to the environment, rather than vice versa.

• Synthetic biology involves creating new cells with artificial DNA made in the laboratory.

• There is public concern about the application of recombinant DNA technology to food production.