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Chapter 17 : The Polymerase Chain Reaction

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The Polymerase Chain Reaction, Page 1 of 2

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Abstract:

The polymerase chain reaction (PCR) has become a key tool in molecular biology research and in biotechnology applications. In the student activity, students use paper models to simulate the steps of the PCR. The model exercise demonstrates how DNA polymerase can be used to make multiple copies of a specific DNA fragment and shows how the technique can be used to detect a specific DNA molecule (such as the chromosome of a disease-causing microorganism) in a sample. Automated DNA sequencing is also based on PCR technology. PCR is a simple technique that combines in vitro DNA synthesis by DNA polymerase and hybridization. To start the reaction, the double-stranded sample DNA in the mixture is denatured into single strands by heating it. The mixture is then cooled so that hybridization of complementary strands can occur. In the next step, the DNA polymerase enzyme synthesizes a second DNA strand on each of the two original strands, using the free nucleotides in the solution. The annealed primer serves as a starting point. New DNA is synthesized from the 3' end of each primer, extending in only one direction. In early PCR methods, new DNA polymerase had to be added after each denaturation step, because the high heat necessary for denaturation destroyed the enzyme. This paper model very accurately demonstrates the steps of PCR and shows how a specific DNA segment can be amplified from a single copy. The second part of the activity illustrates how PCR is used as a diagnostic tool.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17

Key Concept Ranking

Agarose Gel Electrophoresis
0.48266125
Quantitative PCR
0.4766667
DNA Synthesis
0.4502295
0.48266125
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Figures

Image of Figure 17.3
Figure 17.3

The starting template and primers for paper PCR. The colored paper strips are not shown.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Image of Figure 17.4
Figure 17.4

Paper PCR. Primers are hybridized to the denatured template strands.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Image of Figure 17.5
Figure 17.5

Paper PCR, products of the first round of DNA synthesis. Note that the primers are part of the product DNA strands. The base sequence of the product strands has been written on the paper strips.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Image of Figure 17.6
Figure 17.6

Paper PCR, hybridization for the second round of DNA synthesis. It does not matter that the letters on some of the strands are upside down.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Image of Figure 17.7
Figure 17.7

Paper PCR, products of the second round of DNA synthesis.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Download as Powerpoint
Image of Figure 17.8
Figure 17.8

Paper PCR, products of the third round of DNA synthesis. These products include the first molecules of what will be the major product of many rounds of synthesis: the short products extending exactly from one primer sequence to the other. These molecules are the third one down in the left-hand column and the second one down in the right-hand column.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Image of Figure 17.1
Figure 17.1

PCR.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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Image of Figure 17.2
Figure 17.2

Accumulation of the major PCR product with increasing numbers of amplification cycles. The template for this PCR reaction is lambda DNA, and the primers hybridize to it so that the major PCR product is a 1,106-base pair segment. Lane 1 contains DNA fragments from a HindIII digestion of lambda DNA as size markers. Lane 2 shows the PCR mixture before amplification. Lanes 3 to 5 show the PCR mixture after 5, 10, and 15 amplification cycles.

Citation: Kreuzer H, Massey A. 2008. The Polymerase Chain Reaction, p 270-279. In Molecular Biology and Biotechnology: A Guide for Teachers, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816100.ch17
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References

/content/book/10.1128/9781555816100.chap17a
1. Mullis, K. B. 1990. The unusual origin of the polymerase chain reaction. Scientific American 262:5665.
2. Paabo, S. 1993. Ancient DNA. Scientific American 269:8692.
3. Poinar, G. 1999. Ancient DNA. American Scientist 87:446457.

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