1887

Chapter 27 : Nucleic Acid Analysis

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

Category: Microbial Genetics and Molecular Biology

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Preview Magnify
Zoom in
Zoomout

Nucleic Acid Analysis, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap27-1.gif /docserver/preview/fulltext/10.1128/9781555817497/9781555812232_Chap27-2.gif

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
Highlighted Text: Show | Hide
Loading full text...

Full text loading...

Figures

Nucleic Acid Analysis
[com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555817497.chap27-1,webId=/content/author/10.1128/9781555817497.chap27-1,properties={foaf_givenname=William, foaf_name=William Hendrickson, foaf_surname=Hendrickson}], com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555817497.chap27-2,webId=/content/author/10.1128/9781555817497.chap27-2,properties={foaf_givenname=Don, foaf_name=Don Walthers, foaf_surname=Walthers}]]
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.

10.1128/9781555817497/fig27-1_thmb.gif
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
Permissions and Reprints Request Permissions
Download as Powerpoint
/content/10.1128/9781555817497.chap27.fig27-2
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.

10.1128/9781555817497/fig27-2_thmb.gif
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
Permissions and Reprints Request Permissions
Download as Powerpoint
/content/10.1128/9781555817497.chap27.fig27-3
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.

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
Download as Powerpoint
/content/10.1128/9781555817497.chap27.fig27-4
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.

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
Permissions and Reprints Request Permissions
Download as Powerpoint

References

/content/book/10.1128/9781555817497.chap27
1. Ansorge, W.,, and S. Labeit. 1984. Field gradients improve resolution on DNA sequencing gels. J. Biochem. Biophys. Methods 10: 237– 243.
2. Ausubel, F. M.,, R. Brent,, R. E. Kingston,, D. D. Moore,, J. G. Seidman,, J. A. Smith,, and K. Struhl. 2003. Current Protocols in Molecular Biology. Wiley-Interscience, New York, NY.
3. Beloin, C.,, S. McKenna,, and C. J. Dorman. 2002. Molecular dissection of VirB, a key regulator of the virulence cascade of Shigella flexneri. J. Biol. Chem. 277: 15333– 15344.
4. Berk, A. J.,, and P. A. Sharp. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12: 721– 732.
5. Borowiec, J. A.,, L. Zhang,, S. Sasse-Dwight,, and J. D. Gralla. 1987. DNA supercoiling promotes formation of a bent repression loop in lac DNA. J. Mol. Biol. 196: 101– 111.
6. Boucher, P. E.,, A. E. Maris,, M. S. Yang,, and S. Stibitz. 2003. The response regulator BvgA and RNA polymerase alpha subunit C-terminal domain bind simultaneously to different faces of the same segment of promoter DNA. Mol. Cell 11: 163– 173.
7. Brenowitz, M.,, D. F. Senear,, M. A. Shea,, and G. K. Ackers. 1986. Quantitative DNase I footprint titration: a method for studying protein-DNA interactions. Methods Enzymol. 130: 132– 181.
8. Brenowitz, M.,, E. Jamison,, A. Majumdar,, and S. Adhya. 1990. Interaction of the Escherichia coli Gal repressor protein with its DNA operators in vitro. Biochemistry 29: 3374– 3383.
9. Carey, J. 1988. Gel retardation at low pH resolves trp repressor- DNA complexes for quantitative study. Proc. Natl. Acad. Sci. USA 85: 975– 979.
10. Carey, J. 1991. Gel retardation. Methods Enzymol. 208: 103– 117.
11. Chien, A.,, D. B. Edgar,, and J. M. Trela. 1976. Deoxyribonucleic polymerase from the extreme thermophile Thermus aquaticus. J. Bacteriol. 127: 1550– 1557.
12. Crothers, D. M.,, M. R. Gartenberg,, and T. E. Shrader. 1991. DNA bending in protein-DNA complexes. Methods Enzymol. 208: 118– 146.
13. Das, P. M.,, K. Ramachandran,, J. vanWert,, and R. Singal. 2004. Chromatin immunoprecipitation assay. BioTechniques 37: 961– 969.
14. Fried, M.,, and D. M. Crothers. 1981. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9: 6505-– 6525.
15. Fried, M.,, and D. M. Crothers. 1983. CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects. Nucleic Acids Res. 11: 141– 158.
16. Fried, M. G.,, and D. M. Crothers. 1984. Equilibrium studies of the cyclic AMP receptor protein-DNA interaction. J. Mol. Biol. 172: 241– 262.
17. Fried, M. G.,, and D. M. Crothers. 1984. Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J. Mol. Biol. 172: 263– 282.
18. Galas, D. J.,, and A. Schmitz. 1978. DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5: 3157– 3170.
19. Garner, M. M.,, and A. Revzin. 1982. Stoichiometry of catabolite activator protein/adenosine cyclic 3′,5′- monophosphate interactions at the lac promoter of Escherichia coli. Biochemistry 21: 6032– 6036.
20. Grainger, D. C.,, T. A. Belyaeva,, D. J. Lee,, E. I. Hyde,, and S. J. Busby. 2003. Binding of the Escherichia coli MelR protein to the melAB promoter: orientation of MelR subunits and investigation of MelR-DNA contacts. Mol. Microbiol. 48: 335– 348.
21. Grainger, D. C.,, T. A. Belyaeva,, D. J. Lee,, E. I. Hyde,, and S. J. Busby. 2004. Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with the C-terminal domain of the RNA polymerase alpha subunit. Mol. Microbiol. 51: 1311– 1320.
22. Grainger, D. C.,, T. W. Overton,, N. Reppas,, J. T. Wade,, E. Tamai,, J. L. Hobman,, C. Constantinidou,, K. Struhl,, G. Church,, and S. J. Busby. 2004. Genomic studies with Escherichia coli MelR protein: applications of chromatin immunoprecipitation and microarrays. J. Bacteriol. 186: 6938– 6943.
23. Gralla, J. D. 1985. Rapid “footprinting” on supercoiled DNA. Proc. Natl. Acad. Sci. USA 82: 3078– 3081.
24. Greiner, D. P.,, R. Miyake,, J. K. Moran,, A. D. Jones,, T. Negishi,, A. Ishihama,, and C. F. Meares. 1997. Synthesis of the protein cutting reagent iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate and conjugation to cysteine side chains. Bioconjug. Chem. 8: 44– 48.
25. Hayes, J. J.,, and T. D. Tullius. 1989. The missing nucleoside experiment: a new technique to study recognition of DNA by protein. Biochemistry 28: 9521– 9527.
26. Hecht, A.,, and M. Grunstein. 1999. Mapping DNA interaction sites of chromosomal proteins using immunoprecipitation and polymerase chain reaction. Methods Enzymol. 304: 399– 414.
27. Hendrickson, W. 1985. Protein-DNA interactions studied by the gel electrophoresis-DNA binding assay. Bio- Techniques 4: 198– 207.
28. Hendrickson, W.,, and R. Schleif. 1984. Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay. J. Mol. Biol. 178: 611– 628.
29. Hendrickson, W.,, and R. Schleif. 1985. A dimer of AraC protein contacts three adjacent major groove regions of the araI DNA site. Proc. Natl. Acad. Sci. USA 82: 3129– 3133.
30. Herring, C. D.,, M. Raffaelle,, T. E. Allen,, E. I. Kanin,, R. Landick,, A. Z. Ansari,, and B. O. Palsson. 2005. Immobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays. J. Bacteriol. 187: 6166– 6174.
31. Hong, G. F. 1987. The use of DNase I, buffer gradient gel, and 35S label for DNA sequencing. Methods Enzymol. 155: 93– 110.
32. Hudson, J. M.,, and M. G. Fried. 1990. Co-operative interactions between the catabolite gene activator protein and the lac repressor at the lactose promoter. J. Mol. Biol. 214: 381– 396.
33. Innis, M. A.,, K. B. Myambo,, D. H. Gelfand,, and M. A. D. Brow. 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85: 9436– 9440.
34. Khoury, A. M.,, H. S. Nick,, and P. Lu. 1991. In vivo interaction of Escherichia coli lac repressor N-terminal fragments with the lac operator. J. Mol. Biol. 219: 623– 634.
35. Kornblum, J. S.,, S. J. Projan,, S. L. Moghazeh,, and R. P. Novick. 1988. A rapid method to quantitate non-labeled RNA species in bacterial cells. Gene 63: 75– 85.
36. Kuo, M. H.,, and C. D. Allis. 1999. In vivo cross-linking and immunoprecipitation for studying dynamic protein: DNA associations in a chromatin environment. Methods 19: 425– 433.
37. Kuwabara, M.,, and D. S. Sigman. 1987. Footprinting DNA-protein complexes in situ following gel retardation assays using 1,10-phenanthroline-copper ion: Escherichia coli RNA polymerase- lac promoter complexes. Biochemistry 26: 7234– 7238.
38. Laub, M. T.,, S. L. Chen,, L. Shapiro,, and H. H. McAdams. 2002. Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle. Proc. Natl. Acad. Sci. USA 99: 4632– 4637.
39. Lehrach, H.,, D. Diamon,, J. M. Wozney,, and H. Boedtker. 1977. RNA molecular weight determinations by gel electrophoresis under denaturing conditions: a critical examination. Biochemistry 16: 4743– 4749.
40. Lin, D. C.,, and A. D. Grossman. 1998. Identification and characterization of a bacterial chromosome partitioning site. Cell 92: 675– 685.
41. Liu-Johnson, H. N.,, M. R. Gartenberg,, and D. M. Crothers. 1986. The DNA binding domain and bending angle of Escherichia coli CAP protein. Cell 47: 995– 1005.
42. Nick, H.,, and W. Gilbert. 1985. Detection in vivo of protein- DNA interactions within the lac operon of Escherichia coli. Nature 313: 795– 797.
43. O’Neill, L. P.,, and B. M. Turner. 1996. Immunoprecipitation of chromatin. Methods Enzymol. 274: 189– 197.
44. Orlando, V. 2000. Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem. Sci. 25: 99– 104.
45. Pereira, S. L.,, R. A. Grayling,, R. Lurz,, and J. N. Reeve. 1997. Archaeal nucleosomes. Proc. Natl. Acad. Sci. USA 94: 12633– 12637.
46. Revzin, A. 1989. Gel electrophoresis assays for DNAprotein interactions. BioTechniques 7: 346– 355.
47. Rokop, M. E.,, J. M. Auchtung,, and A. D. Grossman. 2004. Control of DNA replication initiation by recruitment of an essential initiation protein to the membrane of Bacillus subtilis. Mol. Microbiol. 52: 1757– 1767.
48. Salzer, W.,, R. F. Gestland,, and A. Bolle. 1967. In vitro synthesis of bacteriophage lysozyme. Nature 215: 588– 591.
49. Sambrook, J.,, E. F. Fritsch,, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
50. Sanger, F.,, S. Nicklen,, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463– 5467.
51. Sasse-Dwight, S.,, and J. D. Gralla. 1988. Probing cooperative DNA-binding in vivo: the lac O1:O3 interaction. J. Mol. Biol. 202: 107– 109.
52. Shanblatt, S. H.,, and A. Revzin. 1983. Two catabolite activator protein molecules bind to the galactose promoter region of Escherichia coli in the presence of RNA polymerase. Proc. Natl. Acad. Sci. USA 80: 1594– 1598.
53. Shanblatt, S. H.,, and A. Revzin. 1984. Kinetics of RNA polymerase-promoter complex formation: effect of nonspecific DNA-protein interactions. Nucleic Acids Res. 12: 5287– 5306.
54. Sheen, J.-Y.,, and S. Brian. 1988. Electrolyte gradient gels for DNA sequencing. BioTechniques 6: 942– 944.
55. Shin, D.,, and E. A. Groisman. 2005. Signal-dependent binding of the response regulators PhoP and PmrA to their target promoters in vivo. J. Biol. Chem. 280: 4089– 4094.
56. Siebenlist, U.,, and W. Gilbert. 1980. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc. Natl. Acad. Sci. USA 77: 122– 126.
57. Solomon, M. J.,, and A. Varshavsky. 1985. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA 82: 6470– 6474.
58. Spencer, V. A.,, J. M. Sun,, L. Li,, and J. R. Davie. 2003. Chromatin immunoprecipitation: a tool for studying histone acetylation and transcription factor binding. Methods 31: 67– 75.
59. Straney, D. C.,, S. B. Straney,, and D. M. Crothers. 1989. Synergy between Escherichia coli CAP protein and RNA polymerase in the lac promoter open complex. J. Mol. Biol. 206: 41– 57.
60. Tabor, S.,, and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82: 1074– 1078.
61. Tabor, S.,, and C. C. Richardson. 1987. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 84: 4767– 4771.
62. Tullius, T. D. 1989. Physical studies of protein-DNA complexes by footprinting. Annu. Rev. Biophys. Biophys. Chem. 18: 213– 237.
63. Tullius, T. D.,, and B. A. Dombroski. 1986. Hydroxyl radical “footprinting”: high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein. Proc. Natl. Acad. Sci. USA 83: 5469– 5473.
64. Tullius, T. D.,, B. A. Dombroski,, M. E. A. Churchill,, and L. Kam. 1987. Hydroxyl radical footprinting: a high resolution method for mapping protein-DNA contacts. Methods Enzymol. 155: 537– 558.
65. Weaver, R. F.,, and C. Weissman. 1979. Mapping of RNA by a modification of the Berk-Sharp procedure: the 5′ termini of 15S β-globin mRNA precursor and mature 10S β- globin mRNA have identical map coordinates. Nucleic Acids Res. 7: 1175– 1193.
66. Wigneshweraraj, S. R.,, A. Ishihama,, and M. Buck. 2001. In vitro roles of invariant helix-turn-helix motif residue R383 in sigma(54) (sigma(N)). Nucleic Acids Res. 29: 1163– 1174.
67. Wu, H. M.,, and D. M. Crothers. 1984. The locus of sequence-directed and protein-induced DNA bending. Nature 308: 509– 513.
68. Yang, C.-C.,, and H. A. Nash. 1989. The interaction of Escherichia coli IHF protein with its specific binding sites. Cell 57: 869– 880.

Tables

Nucleic Acid Analysis
[com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555817497.chap27-1,webId=/content/author/10.1128/9781555817497.chap27-1,properties={foaf_givenname=William, foaf_name=William Hendrickson, foaf_surname=Hendrickson}], com.pub2web.rdf.cci.facet.ContentItem[id=http://asm.metastore.ingenta.com/content/author/10.1128/9781555817497.chap27-2,webId=/content/author/10.1128/9781555817497.chap27-2,properties={foaf_givenname=Don, foaf_name=Don Walthers, foaf_surname=Walthers}]]
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

10.1128/9781555817497/tab27-1_thmb.gif
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

Back to top
This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error