Similarity Analysis of DNAs

John L. Johnson; William B. Whitman

doi:10.1128/9781555817497.ch26

Chapter 26 : Similarity Analysis of DNAs

Authors: John L. Johnson, William B. Whitman

Content Type: Reference

Publication Year: 2007

Category: Microbial Genetics and Molecular Biology

Book DOI: 10.1128/9781555817497

Chapter DOI: 10.1128/9781555817497.ch26

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Abstract:

In contrast to gram-negative bacteria, nearly all gram-positive bacteria must be digested with a lytic enzyme before they can be lysed by a detergent. In addition to lysozyme, which is the enzyme most commonly used, several other enzymes are available. These include N-acetylmuramidase, which is isolated from Streptomyces globisporus and also cleaves the muramic acid backbone; lysostaphin, an endopeptidase that is isolated from Staphylococcus sp. strain K-6-WI and is specific for the cross-linking peptides of other staphylococci. There are two approaches available for disrupting recalcitrant bacteria. The first involves making the cells susceptible to one or more lytic enzymes by growing them in the presence of wall-component analogs or antibiotics, and the second involves the use of any of several physical methods for cell disruption. The moles percent G+C content of DNA can be estimated by several different methods. The methods described in this chapter use thermal denaturation, high-performance liquid chromatography (HPLC), or dye-binding fluorimetry. Bacteriologists have been criticized for being a bit sloppy in doing and reporting DNA reassociation experiments, compared with those researchers working with eucaryotes. Although some of this criticism is justified, there are additional reasons for greater variability in bacterial DNA experiments. First, the sheer number of bacteriology laboratories involved in performing DNA reassociation experiments has contributed to the variability of results. Second, the hydroxylapatite (HA) procedure has been used for most eucaryotic studies, whereas all of the procedures discussed in the chapter have been used extensively by bacteriologists.

Citation: Johnson J, Whitman W. 2007. Similarity Analysis of DNAs, p 624-652. 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.ch26

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Similarity Analysis of DNAs

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FIGURE 1

Spooling DNA onto a glass rod during precipitation by ethanol. Photo by D. Arbour.

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FIGURE 1

Spooling DNA onto a glass rod during precipitation by ethanol. Photo by D. Arbour.

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FIGURE 2

Hyperchromic shift tracings for two samples of DNA in an automatic recording spectrophotometer. The points at which any vertical line crosses the absorbance and temperature curves give values for these two parameters at a given time. The inflection points are estimated at one-half of the hyperchromatic shift, and the temperature at that time is the melting temperature (T m ).

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FIGURE 2

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FIGURE 3

Separation of deoxynucleosides from the ribonucleosides by HPLC for determination of the moles percent G+C. (A) Separation of ribonucleosides from deoxyribonucleosides at 24°C in 12% methanol on a C18 reverse-phase column. The nucleosides were obtained from enzymatic degradation of Methanococcus voltae nucleic acid. (B) Separation of nucleosides under the optimal conditions for resolution of deoxyguanosine (dG) and thymidine (dT), which were 38°C and 12% methanol for this particular column. Under these conditions, guanosine (G) and 5-methyldeoxycytidine (mC) elute as minor peaks just prior to dG and deoxycytidine (dC) and uridine (U) are not resolved. The nucleoside mixture was prepared from standards. (C) Separation of nucleosides from the nucleotide monophosphates. The chromatography was performed as for panel B. The nucleoside mixture contained 2% of the nucleotide monophosphates. Under these conditions, the nucleotides appear as minor peaks near the nucleosides. Possible products of incomplete degradation, the nucleotides can interfer with the determination of dG and dT. Abbreviations: A, adenosine; dA, deoxyadenosine; dAMP, deoxyadenosine monophosphate; C, cytidine; dC, deoxycytidine; mC, 5-methyldeoxycytidine; G, guanosine; dG, deoxyguanosine; dGMP, deoxyguanosine monophosphate; dT, thymidine; U, uridine; X, unidentified compound. From reference 79 .

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FIGURE 3

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FIGURE 4

Specialized equipment for DNA reassociation experiments. (A) Stainless-steel holder for vials, used during incubation in a water bath. (B) Plexiglas rack for setting up experiments. (C) Plexiglas washing chamber for membrane filters. (D) Autoinjection tubes used for iodinating nucleic acids. (E) Polyethylene tubes (0.2 ml) for use in reassociation experiments.

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FIGURE 4

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FIGURE 5

Plexiglas filtration devices for immobilizing DNA on membranes.

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FIGURE 5

Plexiglas filtration devices for immobilizing DNA on membranes.

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FIGURE 6

Diagram of apparatus for testing the thermostability of hybrid duplexes. A piece of 7-mm-diameter glass tubing (A) is sealed with a rubber plug (C), which is made from a rubber stopper by use of a cork borer. Membrane filters (E) and Teflon washers (F) are impaled on an insect pin (D) close to the head of the pin. The pointed end of the pin is inserted into the rubber plug. This assembly is placed into a 13- by 100-mm glass test tube (B) containing 1.2 ml of 0.5× SSC. The test tube is then placed in a heated-water bath.

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FIGURE 6

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FIGURE 8

Scheme for performing free-solution DNA reassociation experiments, assayed either by the HA or S1 nuclease procedure. TCA, trichloroacetic acid.

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FIGURE 8

Scheme for performing free-solution DNA reassociation experiments, assayed either by the HA or S1 nuclease procedure. TCA, trichloroacetic acid.

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FIGURE 7

Tube rack for thermal stability experiments. This rack fits into the opening of a circulating-water bath. The small amount of exposed surface area cuts down on evaporation.

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FIGURE 7

Tube rack for thermal stability experiments. This rack fits into the opening of a circulating-water bath. The small amount of exposed surface area cuts down on evaporation.

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FIGURE 9

Thermal stability profiles obtained with the hypothetical HA procedure data in Table 2 . Symbols: ∇, percent counts per minute eluted at each temperature for heterologous duplexes; ▴, sum of percent counts per minute eluted at each temperature for heterologous duplexes; ×, percent counts per minute eluted at each temperature for homologous duplexes; +, sum of percent counts per minute eluted at each temperature for homologous duplexes.

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FIGURE 9

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FIGURE 10

Generalized C 0 t plot. At C 0 t values of less than or equal to a, 1/(1 + C 0 t) values are 0.9 or greater. At C 0 t values equal to or greater than b, 1/(1 + C 0 t) values are less than 0.1.

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FIGURE 10

Generalized C 0 t plot. At C 0 t values of less than or equal to a, 1/(1 + C 0 t) values are 0.9 or greater. At C 0 t values equal to or greater than b, 1/(1 + C 0 t) values are less than 0.1.

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FIGURE 11

Scheme for performing membrane DNA reassociation experiments by using either the direct-binding or competition procedure.

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FIGURE 11

Scheme for performing membrane DNA reassociation experiments by using either the direct-binding or competition procedure.

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FIGURE 12

Apparatus for counting radioactivity on membrane filters. A, scintillation vial; B, insect pin; C, nonaqueous- based cocktail; D, duplicate filters impaled on pin.

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FIGURE 12

Apparatus for counting radioactivity on membrane filters. A, scintillation vial; B, insect pin; C, nonaqueous- based cocktail; D, duplicate filters impaled on pin.

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FIGURE 13

Comparison of S1 and HA reassociation methods at different temperatures. Symbols: ▄, S1 method, 75°C; *, HA method, 75°C; +, S1 method, 60°C; □, HA method, 60°C.

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FIGURE 13

Comparison of S1 and HA reassociation methods at different temperatures. Symbols: ▄, S1 method, 75°C; *, HA method, 75°C; +, S1 method, 60°C; □, HA method, 60°C.

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Tables

Similarity Analysis of DNAs

/content/10.1128/9781555817497.chap26.tab26-1

TABLE 1

Hypothetical examples of the calculations of percent similarity values in the free-solution method by the HA and S1 nuclease procedures

10.1128/9781555817497/tab26-1_thmb.gif

10.1128/9781555817497/tab26-1.gif

TABLE 1

Hypothetical examples of the calculations of percent similarity values in the free-solution method by the HA and S1 nuclease procedures

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TABLE 2

Calculations for hypothetical data from HA thermal stability profile

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10.1128/9781555817497/tab26-2.gif

TABLE 2

Calculations for hypothetical data from HA thermal stability profile

/content/10.1128/9781555817497.chap26.tab26-3

TABLE 3

C0t values for various DNA concentrations and reassociation times

a Equivalents: 1 μg/110 μl = 9.1 μg/ml or 0.27 × 10−4 mol/liter; 5 μg/110 μl = 45 μg/ml or 1.37 × 10−4 mol/liter; 20 μg/110 μl = 182 μg/ml or 5.49 × 10−4 mol/liter; 30 μg/110 μl = 272 μg/ml or 8.24 x 10−4 mol/liter.

10.1128/9781555817497/tab26-3_thmb.gif

10.1128/9781555817497/tab26-3.gif

TABLE 3

C0t values for various DNA concentrations and reassociation times