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Chapter 27 : Nucleic Acid Analysis

Authors: William Hendrickson, Don Walthers
Content Type: Reference
Publication Year: 2007

Category: Microbial Genetics and Molecular Biology

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Abstract:

This chapter describes several of the most common nucleic acid analyses performed in vitro to characterize a cloned DNA segment carrying a gene or genes. Newer polymerases that are used for DNA sequencing include modified T7 phagederived DNA polymerase and a variety of thermostable DNA polymerases such as that from the thermophilic bacterium . The authors have successfully used the kit marketed by U.S. Biochemicals for many years; however, other products may be just as effective. It is strongly recommended that laboratories use kits from U.S. Biochemicals or competing manufacturers for applications in which they need to do manual sequencing and gel electrophoresis. The gel mobility shift assay (also called the gel retardation assay) is based on the differences in the degrees of electrophoretic mobility between nucleic acid fragments and nucleic acid-protein complexes. Although the assay has been used successfully to study binding to RNA, this discussion will be limited to the most common use, the study of binding to dsDNA. The outcome of the assay is the identification of specific DNA targets of the protein of interest. A section reviews ChIP assays from a number of laboratories relevant to prokaryotic systems. Commonly used in vitro techniques described elsewhere in the chapter generally require biochemical purification of a DNA-binding protein of interest and some knowledge concerning the location of its DNA target(s). Once a gene or promoter has been cloned, it is of interest to determine the start site and the size of the RNA transcript.

Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27

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Nucleic Acid Analysis
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Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27
/content/10.1128/9781555817497.chap27.fig27-1
FIGURE 1

Autoradiogram of a sequencing gel. A central portion of the gel approximately 100 bases from the primer is shown. Lanes are identified at the top, and a portion of the sequence is indicated at the side.

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10.1128/9781555817497/fig27-1.gif
Image of FIGURE 1
FIGURE 1

Autoradiogram of a sequencing gel. A central portion of the gel approximately 100 bases from the primer is shown. Lanes are identified at the top, and a portion of the sequence is indicated at the side.

Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27
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FIGURE 2

Gel mobility shift assay. Shown is an autoradiogram from an assay with a 200-bp DNA fragment corresponding to 500 cpm per sample run on a 6% polyacrylamide gel for 1 h at 8 V/cm. Lanes 2 through 5 contain samples with three-fold increases in levels of protein (purified AraC protein). Film was exposed overnight at –70°C with an intensifying screen.

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10.1128/9781555817497/fig27-2.gif
Image of FIGURE 2
FIGURE 2

Gel mobility shift assay. Shown is an autoradiogram from an assay with a 200-bp DNA fragment corresponding to 500 cpm per sample run on a 6% polyacrylamide gel for 1 h at 8 V/cm. Lanes 2 through 5 contain samples with three-fold increases in levels of protein (purified AraC protein). Film was exposed overnight at –70°C with an intensifying screen.

Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27
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FIGURE 3

DNase I footprint. Shown is an autoradiogram from a footprinting assay of AraC protein binding to a synthetic binding site cloned into the promoter. G+A and A+C chemical sequencing reactions ( ) were run in the first two lanes. 0, reactions with no protein; +, reactions with a twofold excess of protein relative to DNA. Each sample was loaded onto an 8% sequencing gel in an amount corresponding to 5,000 cpm. The protected region is indicated by a bracket. Two bands of strongly enhanced cleavage appear within the protected region.

10.1128/9781555817497/fig27-3_thmb.gif
10.1128/9781555817497/fig27-3.gif
Image of FIGURE 3
FIGURE 3

DNase I footprint. Shown is an autoradiogram from a footprinting assay of AraC protein binding to a synthetic binding site cloned into the promoter. G+A and A+C chemical sequencing reactions ( ) were run in the first two lanes. 0, reactions with no protein; +, reactions with a twofold excess of protein relative to DNA. Each sample was loaded onto an 8% sequencing gel in an amount corresponding to 5,000 cpm. The protected region is indicated by a bracket. Two bands of strongly enhanced cleavage appear within the protected region.

Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27
Permissions and Reprints Request Permissions
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FIGURE 4

S1 and primer extension assays. Cross-hatched line, mRNA; solid line, DNA; circle, labeled 5′ end of DNA. The cartoon of a sequencing gel shows sequencing reactions in the center, an S1 reaction to the left, and primer extension to the right.

10.1128/9781555817497/fig27-4_thmb.gif
10.1128/9781555817497/fig27-4.gif
Image of FIGURE 4
FIGURE 4

S1 and primer extension assays. Cross-hatched line, mRNA; solid line, DNA; circle, labeled 5′ end of DNA. The cartoon of a sequencing gel shows sequencing reactions in the center, an S1 reaction to the left, and primer extension to the right.

Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27
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Tables

Nucleic Acid Analysis
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Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27
/content/10.1128/9781555817497.chap27.tab27-1
TABLE 1

Preparation of buffers and solutions

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10.1128/9781555817497/tab27-1.gif
Generic image for table
TABLE 1

Preparation of buffers and solutions

Citation: Hendrickson W, Walthers D. 2007. Nucleic Acid Analysis, p 653-675. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch27

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