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Chapter 5 : Recombinant DNA Technology

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Abstract:

This chapter focuses on some of the tools scientists use in carrying out research one reads about in headlines. It is a conceptual guide to basic procedures that are used over and over again in biotechnology. It focuses on some of the enzymes and other fundamental tools for working with DNA and cells that are used to manipulate and analyze DNA, including determining its sequence; to clone DNA; and to analyze proteins. It talks about manipulating and analyzing DNA. Just as there are many kinds of cloning vectors, there are many ways to introduce DNA into a host cell. Regardless of the vector and transfer method used, once the recombinant DNA has been introduced into a batch of host cells, the next task is to identify those cells that took up and are maintaining the recombinant DNA. cDNA libraries are based on mRNA from the starting tissue, so they represent only those genes being actively transcribed. To make monoclonal antibodies to a specific protein or other antigen, researchers inoculate a mouse with that substance. To determine whether the fragment of interest is present in the vector DNA, additional analysis, such as hybridization to a probe matching the sequence of interest, PCR with appropriate primers, or purification and sequencing of the recombinant plasmid, is usually performed. The cloned gene is manipulated in the laboratory using the tools and techniques described above to get it ready for insertion into the target organism. Finally, the chapter discusses genetic engineering of plants and animals.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5

Key Concept Ranking

Restriction Fragment Length Polymorphism
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Figures

Image of Figure 5.1
Figure 5.1

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. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.2
Figure 5.2

Gel electrophoresis separates DNA fragments by size.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.3
Figure 5.3

A 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. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.4
Figure 5.4

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

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.5
Figure 5.5

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. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.6
Figure 5.6

Hybridization analysis. A single-stranded probe is added to denatured sample DNA.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.7
Figure 5.7

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 on the membrane.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.8
Figure 5.8

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. The primer becomes part of the new DNA molecule.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.9
Figure 5.9

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. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.10
Figure 5.10

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. The primers are usually synthetic oligonucleotides.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.11
Figure 5.11

Testing for the presence of a DNA sequence using PCR.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.12
Figure 5.12

Cloning of DNA in recombinant bacterial plasmids.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.13
Figure 5.13

DNA libraries.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.14
Figure 5.14

A home pregnancy test uses antibodies to detect the pregnancy hormone, HCG. (A) The absorbent wick contains dispersed antibody molecules labeled with a colored indicator. (B) To perform the test, the wick is dipped in urine, which migrates up the absorbent material. (C) If the urine contains HCG, the dispersed colored antibody molecules will bind to it and be swept along with the urine. (D) 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 is a positive result.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.15
Figure 5.15

Isolating mutants that cannot biosynthesizehistidine (His).

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.16
Figure 5.16

Using genetics to find an gene for histidine biosynthesis.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.17
Figure 5.17

Genetic testing for SSA. bp, base pairs.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.18
Figure 5.18

An evolutionary tree based on the amino acid sequence of the protein cytochrome The numbers represent the numbers of amino acid changes between two nodes of the tree. For example, when the common ancestor of cartilaginous fish diverged from the line leading to bony fish, mammals, etc., it evolved and accumulated two amino acid changes in its cytochrome protein before the lines leading to dogfish and lamprey diverged. The dogfish line accumulated nine more amino acid changes in becoming the modern organism; the lamprey accumulated eight changes. Thus, the amino acid sequences of cytochrome in lamprey and dogfish have 17 differences. (From A. Lehninger, D. Nelson, and M. Cox, 2nd ed., Worth Publishers, Inc., New York, NY, 1993.)

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.19
Figure 5.19

Neanderthals (skull cast on right), with their large brains and anatomy similar to ours, were long believed to be direct ancestors of modern humans (skull model on left). Mitochondrial DNA analysis suggests that modern humans instead arose independently from Neanderthals and displaced them. (Photograph courtesy of SOMSO Models, Coburg, Germany.)

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.20
Figure 5.20

DNA fingerprinting by RFLP analysis. Radioactive probes are detected by exposing the membrane to X-ray film after hybridization is complete.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.21
Figure 5.21

Genes are often cloned so that their expression can be controlled. For example, a gene cloned with the promoter will be expressed only if lactose is present in the growth medium.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.22
Figure 5.22

Plant tissue culture. (A) Pieces of plant tissue can be induced to form undifferentiated callus cells (plates on right). Manipulation of hormones in the growth medium causes the callus to differentiate into tiny plantlets (plates on left). (B) Close-up of plantlet. Note the fuzzy roots and tiny leaves. (Photographs courtesy of Syngenta.)

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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Image of Figure 5.23
Figure 5.23

Making a transgenic mouse with ES cells.

Citation: Kreuzer H, Massey A. 2008. Recombinant DNA Technology, p 137-168. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch5
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References

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