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Section 3 : DNA Cloning

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Abstract:

This section talks about exercises of DNA cloning by polymerase chain reaction (PCR), and for protein expression. Cloning a new piece of DNA has almost countless uses. These uses range from determining the exact nucleotide sequence of a gene or piece of DNA to obtaining the protein product of a gene for use as a medication to gene therapy. To clone a piece of DNA, one must have a desired piece of DNA to clone. This may involve finding a gene that a researcher only suspects exists. Sometimes cloning a piece of DNA may simply involve moving a gene from one plasmid or vector to a different vector that has special properties. Polymerase chain reaction (PCR) may be used to clone desired fragments of DNA from complex genomes. Evolutionary relationships may be studied by using PCR. DNA cloning provides a mechanism for producing large quantities of insert DNA, which may be used for DNA sequencing or as a probe in Northern or Southern blotting. A gene's ultimate role, however, is protein production. Understanding primary DNA sequence is only one part of elucidating protein function. Gene sequences, cloned into special protein expression vectors, may be used to produce the actual protein for study or therapeutic use. Biotechnologists may even mutate DNA sequences, recombinantly produce an altered form of the protein, and study the function of particular domains of the protein. The section also includes exercises on DNA ligation and cloning, DNA cloning by polymerase chain reaction, and DNA cloning for protein expression.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3

Key Concept Ranking

Agarose Gel Electrophoresis
0.4440821
Plasma Membrane
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DNA Restriction Enzymes
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Figures

Image of Figure 13.1
Figure 13.1

The mechanism of blue or white color selection to screen for plasmids that contain insert DNA. (Left) IPTG induces the operon, turning on the gene that produces β-Gal. X-Gal, present in the bacterial growth medium, is cleaved by β-Gal, and bacterial colonies turn blue. (Right) A DNA fragment, inserted into the multiple cloning site (MCS) of the plasmid, disrupts the gene, and no (β-Gal is produced. In this case, X-Gal remains intact and bacterial colonies are white, indicating the presence of plasmids containing inserts in these bacteria.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Image of Figure 13.2
Figure 13.2

The Cloning vector pGEM-3Zf+.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Image of Figure 13.3
Figure 13.3

Plasmid pHGH107, containing the gene for hGH.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 13.4

The nucleotide sequence of the hGH gene.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 14.1

pGEM-T-Easy vector. This vector (Promega) contains single T overhangs for cloning of PCR products with A-nucleotide overhangs.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 14.2

Summary of PCR cloning.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 15.1

A generic prokaryotic expression vector.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 15.2

Test for production of amylase, indicating degradation of starch. Bacteria are plated onto starch agar and incubated for 48 hours. The plates are flooded with IKI solution. Amylase-positive bacteria are on the left, negative on the right.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 15.3

A two-step expression vector system utilizing elements of the T7 bacteriophage.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 15.4

The nucleotide sequence of the mouse amylase gene.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Figure 15.5

The amino acid reading frames of pGEMEX-1 and pGEMEX-2. (A) The pCEMEX-1 vector DNA sequence, indicating reading frames for amino acids. (B) The pCEMEX-2 vector DNA sequence, indicating reading frames for amino acids. The two boldface G nucleotides indicate additions to the sequence, causing a shift in the reading frame.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
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References

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Tables

Generic image for table
Table 14.1

Percent difference in hemoglobins of selected vertebrates compared with humans

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
Generic image for table
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dNTPs, deoxynucleoside triphosphates.

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3
Generic image for table
Untitled

Citation: Scheppler J, Cassin P, Gambier R. 2000. DNA Cloning, p 99-139. In Biotechnology Explorations. ASM Press, Washington, DC. doi: 10.1128/9781555818135.ch3

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