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Chapter 18 : DNA Sequencing: The Terminators

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DNA Sequencing: The Terminators, Page 1 of 2

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Abstract:

Determining the base sequence of a piece of DNA is a critical step in applications of biotechnology. In DNA replication, a nucleotide complementary to the template base is brought into position. The DNA polymerase then adds it to the growing DNA strand by forming a bond between the 5' phosphate group of the new nucleotide and the 3' OH group of the previous nucleotide. The chain terminators used in DNA sequencing are the dideoxynucleotides. The newly synthesized molecules are separated by size and visualized by exposing the gel to photographic film. The A reaction lane shows bands that correspond in length to the site of each T in the template. The G reaction lane shows bands whose lengths correspond to the site of template C’s, and so on. The sequence of the new DNA strand is “read” from the sequencing gel by starting at the bottom (the shortest new molecule) and reading upward. As in PCR, the mixture is put through temperature cycles of denaturation, hybridization, and DNA synthesis. Unlike a PCR, only one primer is present, so DNA synthesis occurs using only one strand of the parental DNA as a template. The AIDS virus encodes a special enzyme, reverse transcriptase, that synthesizes DNA using the viral RNA as a template. HIV infects cells as an RNA genome packaged with the enzymes reverse transcriptase and integrase inside a protein envelope. The herpesviruses are DNA viruses with relatively large genomes. Herpesvirus diseases can now be treated with the chain terminator acyclovir.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing: The Terminators, p 214-220. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch18

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DNA Synthesis
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Figures

Image of Figure 18.1
Figure 18.1

Normal deoxynucleoside shown with chain terminators. All are incorporated into DNA from their triphosphate forms.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing: The Terminators, p 214-220. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch18
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Image of Figure 18.2
Figure 18.2

DNA replication. Base pairing between an incoming deoxynucleoside triphosphate and the template strand of DNA guides the formation of a new complementary strand.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing: The Terminators, p 214-220. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch18
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Image of Figure 18.3
Figure 18.3

A student is loading a sequencing gel. A sequencing gel is what is called a vertical gel—the DNA runs from top to bottom. It is tall and quite thin: less than 1 millimeter thick. Sequencing gels are made with the substance acrylamide instead of agarose. Acrylamide forms a much tighter mesh than agarose, enabling the separation of DNA molecules that are only 1 base pair different in length.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing: The Terminators, p 214-220. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch18
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Image of Figure 18.4
Figure 18.4

Autoradiogram of a sequencing gel. The scientist who did this sequencing procedure was screening several plasmids from transformants to see if she got a clone she was trying to construct. She did. In fact, every one of the plasmids had the sequence she wanted.

Citation: Kreuzer H, Massey A. 2008. DNA Sequencing: The Terminators, p 214-220. In Molecular Biology and Biotechnology: A Guide for Students, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817480_ch18
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