CHAPTER 17 Recombinant DNA and Biotechnology

Chapter 17: Recombinant DNA and Biotechnology Cleaving and Rejoining DNA Cloning Genes Sources of Genes for Cloning Some Additional Tools for DNA Manipulation Biotechnology: Applications of DNA Manipulation

Cleaving and Rejoining DNA Knowledge of DNA transcription, translation, and replication has been used to create recombinant DNA molecules, made up of sequences from different organisms. 3

Cleaving and Rejoining DNA Restriction enzymes, which are made by microbes as a defense mechanism against viruses, bind to DNA at specific sequences and cut it. Review Figure 17.1 4

figure 17-01.jpg 17.1 Figure 17.1

Cleaving and Rejoining DNA DNA fragments generated from cleavage by restriction enzymes can be separated by size using gel electrophoresis. The fragments' sequences can be further identified by hybridization with a probe. Review Figures 17.2, 17.3 6

figure 17-02.jpg 17.2 Figure 17.2

figure 17-03.jpg 17.3 Figure 17.3

Cleaving and Rejoining DNA Many restriction enzymes make staggered cuts in the two strands of DNA, creating “sticky ends” with unpaired bases. The sticky ends can be used to create recombinant DNA if DNA molecules from different species are cut with the same restriction enzyme. Review Figure 17.4 9

figure 17-04.jpg 17.4 Figure 17.4

Cloning Genes Bacteria, yeasts, and cultured plant cells are commonly used as hosts for recombinant DNA experiments. 11

Cloning Genes Newly introduced DNA must be part of a replicon if it is to be propagated in host cells. One way to assure this is to introduce it as part of a carrier DNA, or vector, that has a replicon. 12

Cloning Genes Specialized vectors transfect bacteria, yeasts, and plant cells. These must contain a replicon, recognition sequences for restriction enzymes, and genetic markers to identify their presence in host cells. Review Figure 17.5 13

figure 17-05.jpg 17.5 Figure 17.5

Cloning Genes Naked DNA may be introduced into a host cell by chemical or mechanical means. In this case, the DNA must integrate into the host DNA by itself. 15

Cloning Genes When vectors carrying recombinant DNA are incubated with host cells, nutritional, antibiotic resistance, or fluorescent markers can identify which cells contain the vector. Review Figure 17.6 16

figure 17-06a.jpg 17.6 Figure 17.6 – Part 1

figure 17-06b.jpg 17.6 Figure 17.6 – Part 2

Sources of Genes for Cloning The cutting of DNA by a restriction enzyme produces many fragments that can be individually and randomly combined with a vector and inserted into a host to create a gene library. Review Figure 17.8 19

figure 17-08.jpg 17.8 Figure 17.8

Sources of Genes for Cloning The mRNA’s produced in a certain tissue at a certain time can be extracted and used to create complementary DNA (cDNA) by reverse transcription. This cDNA is then used to make a library. Review Figure 17.9 21

figure 17-09.jpg 17.9 Figure 17.9

Sources of Genes for Cloning A third source of DNA is synthetic DNA made in the laboratory. The methods of organic chemistry can be used to create specific, mutated DNA sequences. 23

Some Additional Tools for DNA Manipulation Homologous recombination can be used to “knock out” a gene in an organism. Review Figure 17.10 24

figure 17-10a.jpg 17.10 Figure 17.10 – Part 1

figure 17-10b.jpg 17.10 Figure 17.10 – Part 2

Some Additional Tools for DNA Manipulation DNA chip technology permits the screening of thousands of sequences at the same time. Review Figure 17.11 27

figure 17-11.jpg 17.11 Figure 17.11

Some Additional Tools for DNA Manipulation An antisense RNA complementary to a specific mRNA can prevent its translation by hybridizing to the mRNA. Review Figure 17.12 29

figure 17-12.jpg 17.12 Figure 17.12

Biotechnology: Applications of DNA Manipulation The ability to clone genes has made possible many new applications of biotechnology, such as the large-scale production of eukaryotic gene products. 31

Biotechnology: Applications of DNA Manipulation For a vector carrying a gene of interest to be expressed in a host cell, the gene must be adjacent to appropriate sequences for its transcription and translation in the host cell. Review Figure 17.13 32

figure 17-13.jpg 17.13 Figure 17.13

Biotechnology: Applications of DNA Manipulation Recombinant DNA and expression vectors have been used to make medically useful proteins that would otherwise have been difficult to obtain in necessary quantities. Review Figure 17.14, Table 17.1 34

figure 17-14a.jpg 17.14 Figure 17.14 – Part 1

figure 17-14b.jpg 17.14 Figure 17.14 – Part 2

table 17-1.jpg 17.1 Table 17.1

Biotechnology: Applications of DNA Manipulation Because plant cells can be cloned to produce adult plants, introduction of new genes into plants via vectors has been advancing rapidly. The result is crop plants carrying new, useful genes. Review Table 17.2 38

table 17-2.jpg 17.2 Table 17.2

Biotechnology: Applications of DNA Manipulation “Pharming” uses transgenic dairy animals that produce useful products in their milk. Review Figure 17.15 40

figure 17-15.jpg 17.15 Figure 17.15

Biotechnology: Applications of DNA Manipulation There is public concern about the applications of biotechnology to food production. 42

Biotechnology: Applications of DNA Manipulation Because the DNA of an individual is unique, the polymerase chain reaction can be used to identify an organism from a small sample of its cells, and to create a DNA fingerprint. Review Figures 17.17, 17.18 43

figure 17-17.jpg 17.17 Figure 17.17

figure 17-18.jpg 17.18 Figure 17.18