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Chapter 15 : The Biotechnology Toolbox

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The Biotechnology Toolbox, Page 1 of 2

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Abstract:

This chapter highlights at some of the enzymes and other fundamental tools for manipulating DNA and cells that enable us to analyze DNA, including determining its sequence, clone DNA, analyze proteins. It focuses on tools and techniques of biotechnology that are based on natural processes and cellular enzymes. The enzymes used by biotechnologists to manipulate DNA are the same enzymes cells use to cut, paste, and copy DNA. Restriction enzymes cut the phosphodiester backbone of DNA molecules at specific base sequences. DNA ligase seals properly aligned DNA fragments together by forming new phosphodiester bonds. Hybridization between a DNA molecule and a labeled probe is used to indicate the presence of the DNA sequence in the probe. Polymerase chain reaction (PCR) amplifies a defined segment of a DNA molecule through the use of specific primers and a polymerase enzyme. PCR can be used to generate many copies of a fragment for cloning or as a detection method. A specific piece of DNA is cloned by introducing it into a host cell that replicates the DNA as it reproduces, generating many identical copies of the DNA molecule. A complementary DNA (cDNA) library is a special type of DNA library in which the cloned DNA fragments are cDNA copies of mRNA taken from a eukaryotic cell. Cell fusion is used to make cells that produce monoclonal antibodies. The complex, high-technology techniques of X-ray diffraction and nuclear magnetic resonance (NMR) are used to determine the structures of proteins.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15

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Figures

Image of Figure 15.1
Figure 15.1

Restriction endonucleases recognize and cut specific sites in a DNA molecule. The arrows indicate the cleavage sites of one such endonuclease.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.2
Figure 15.2

The recognition sites of restriction enzymes are usually palindromic. One of the two identical enzyme subunits cleaves at the same position in the palindrome on each strand. The cleavage sites can be opposite one another, as in Figure 15.1 . When the cleavage sites are staggered, as shown here, cleavage generates identical complementary protruding single strands at the end of every restriction fragment.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.3
Figure 15.3

Gel electrophoresis separates DNA fragments by size.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.4
Figure 15.4

Stained agarose gel showing separated DNA fragments. The outlines of the sample wells are visible at the top of the gel. The lanes at the far right and left contain a mixture of fragments of known size.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.5
Figure 15.5

DNA ligase joins DNA fragments by forming bonds between the 3′ and 5′ ends of the two backbones

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.6
Figure 15.6

Hybridization is the formation of base pairs between two complementary single-stranded nucleic acid molecules. The molecules can be the same or different lengths.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.7
Figure 15.7

Hybridization analysis. A single- stranded probe is added to denatured sample DNA. If the sample DNA contains the base sequence complementary to the probe sequence, the probe will hybridize to the sample and physically stick to it.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.8
Figure 15.8

A given DNA sequence can be localized to a specific restriction fragment by blotting DNA fragments from an electrophoresis gel to a membrane and conducting hybridization analysis of the membrane.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.9
Figure 15.9

DNA polymerase makes copies of existing DNA molecules (known as the template). The DNA synthesis reaction requires a primer hybridized to single-stranded template DNA, the enzyme, and all four nucleotides. DNA polymerase synthesizes a new complementary DNA strand (shown in red) by adding nucleotides to the 3′ end of the primer. The primer thus becomes part of the new DNA molecule.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.10
Figure 15.10

Reverse transcriptase uses RNA as a template and synthesizes a cDNA copy. Through additional reactions, the RNA can be removed and replaced with a second DNA strand.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.11
Figure 15.11

PCR produces many copies of a DNA segment lying between and including the sequences at which two single-stranded primers hybridize to the template DNA molecule.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.12
Figure 15.12

The vivid yellow, orange, and brown colors at the edges of this steaming hot spring in Yellowstone National Park reflect the presence of heat-tolerant microorganisms. One such bacterium from the park is the source of the DNA polymerase used in PCR. (Photograph courtesy of Thomas A.Martin.)

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.13
Figure 15.13

Testing for the presence of a DNA sequence by using PCR.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.14
Figure 15.14

Terminator nucleotides. A 3′ OH group (highlighted) is required for DNA synthesis, because a new nucleotide is added to the existing strand by the formation of a bond between the OH group and the 5′ phosphate group of the incoming nucleotide. A dideoxynucleotide terminates DNA synthesis because it lacks a 3′ OH group.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.15
Figure 15.15

DNA sequencing with DNA polymerase and dideoxynucleotides (ddG, ddA, ddC, and ddT). Four reactions are performed, each one containing a separate dideoxynucleotide. The products of each reaction are separated in adjacent lanes of a gel. DNA sequencing gel. Each set of four lanes represents one sample (four reactions).

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.16
Figure 15.16

The Ti plasmid of is an important cloning vector used in plants. When infects a plant cell, proteins encoded by genes carried on the Ti plasmid transfer the DNA between the T-DNA borders into the plant chromosome. Scientists remove the tumor formation genes from the T-DNA and replace it with DNA to be inserted into the plant genome.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.17
Figure 15.17

Cloning of DNA in recombinant bacterial plasmids.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.18
Figure 15.18

DNA libraries. Genomic DNA library. The insertions in the recombinant plasmids represent the entire DNA content of the organism. cDNA library. The insertions in the recombinant plasmids represent genes that were being expressed in the sample.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.19
Figure 15.19

Fluorescent antibody staining reveals the locations of Hunchback and Kruppel proteins in an early embryo. The Hunchback antibody was labeled with a dye that appears green under fluorescent light, while the Kruppel antibody appears red. When both antibodies are present, the red and green fluorescences combine to make yellow. (Photograph courtesy of Jim Langeland, Steve Paddock, and Sean Carroll, Howard Hughes Medical Institute, University of Wisconsin.)

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.20
Figure 15.20

Fluorescent staining reveals cellular architecture. Antibodies to proteins that form two different kinds of filaments reveal the cytoskeleton, a network that gives a cell its shape. (Photograph courtesy of Nasser M. Rusan.) Epithelial cells in anaphase were stained with an antibody to a spindle fiber protein (green) and a dye that binds to DNA (red), revealing the chromosomes' migration toward what will become new nuclei. (Photograph courtesy of Nasser M. Rusan, 2003 Nikon Small World Contest.)

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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Image of Figure 15.21
Figure 15.21

Home pregnancy tests use antibodies to detect the pregnancy hormone HCG. The absorbent wick contains dispersed antibody molecules labeled with a colored indicator. To perform the test, the wick is dipped in urine, which migrates up the absorbent material. If the urine contains HCG, the dispersed colored antibody molecules will bind to it and be swept along with the urine. The indicator window sits over a line of molecular traps for the antibody. If HCG-antibody complexes are carried up the wick in the urine, they will be trapped there, forming a colored line under the window. The colored line indicates the presence of HCG and a positive result.

Citation: Kreuzer H, Massey A. 2005. The Biotechnology Toolbox, p 359-384. In Biology and Biotechnology. ASM Press, Washington, DC. doi: 10.1128/9781555816094.ch15
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