A laboratory exercise in comparative DNA analysis

Authors


Abstract

This report describes a laboratory exercise that is used in an upper level biochemistry course at a small liberal arts college. The laboratory exercise was designed with three major learning objectives in mind. The first objective is that students learn how to isolate and purify plasmid DNA. The second is that they understand this process on a molecular level. Last, they analyze their DNA samples using a variety of biochemical and biophysical methods. By comparing and contrasting the results, they are to draw conclusions about which methods would be best under a given set of experimental conditions.

One of the main purposes of this exercise is to help students gain practical experience as well as a theoretical understanding of two very common procedures carried out in a biochemistry and/or molecular biology laboratory setting. Isolation and purification of recombinant plasmid DNA, commonly referred to as a “plasmid prep” is a quite common procedure that is a first step in projects ranging from sequencing to protein expression and purification. Although there are a variety of methods that can be used to isolate plasmid DNA (cesium chloride, boiling lysis, and lithium chloride preps, for example) many of today's commercial kits and automated methods are based upon the alkaline lysis technique [1]. Students used an adaptation of the original rapid alkaline extraction procedure of Birnboim and Doly in conjunction with ethanol precipitation to isolate and purify plasmid DNA from bacterial cells. The advantages of this system are that it is extremely inexpensive, is easy to adapt for larger classes, and does not generate a great deal of hazardous or solid waste.

One of the most common types of analyses performed in biochemistry and molecular biology is the quantitation of DNA and assessment of its purity. Students were presented with several options for analysis and assessment of purity of their plasmid DNA samples: UV-visible spectroscopy, fluorometry, and gel electrophoresis. They were asked to compare and contrast the different analytical methods and discuss why the results may have differed. From this they should have been able to conclude that certain analytical methods yield more accurate results under a given set of experimental circumstances.

This laboratory exercise is designed for students in upper level biochemistry and/or molecular biology courses. It is assumed that students have had general and organic chemistry courses as well as an introductory cell biology course. A main goal is to have them integrate knowledge from these disciplines and use it to explain and understand technical procedures on a molecular level. Although there is no text associated with this laboratory, any of the following common texts/lab manuals can be used as references: Fundamentals of Biochemistry [2], Biochemistry, 5th ed. [3], Biochemistry, 3rd ed. [4], and Experiments in Biochemistry [5].

EXPERIMENTAL DESIGN

Materials/Equipment—

The following major equipment is required for this laboratory exercise.

  • UV-visible spectrophotometer

  • Luminescence spectrophotometer

  • Clinical centrifuge

  • Microcentrifuge

  • Horizontal gel electrophoresis apparatus and power supply

  • UV transilluminator

The particular luminescence spectrophotometer used in our laboratory exercise was a QuantaMaster Fluorescence spectrometer (Photon Technology International). It contains both excitation and emission monochromators, although instruments with a single excitation monochromator and filters to monitor emission would work just as well for this particular application. Clear-sided methacrylate cuvettes were used for the fluorescence assays.

All reagents required for making buffers and lysis and electrophoresis solutions were obtained from Sigma. The fluorescent dye Hoechst 33258 was also obtained from Sigma. Protein quantitation was performed using the commercial RC DC protein assay reagents from Bio-Rad. It is important to use protein assay reagents that are compatible with detergents/reducing agents and can be adapted to a microscale assay. DNA molecular weight standards (1-kb ladder) were obtained from New England Biolabs.

Experiment—

The pUC 18 plasmid and either chemically competent or electroporation-competent cells can be obtained from a variety of commercial sources; high efficiency transformation can be achieved by following the supplied protocols. A single colony of pUC 18-transformed bacteria was inoculated into 10 ml of LB broth containing the appropriate antibiotic and grown for 12–20 h with agitation at 37 °C. Plasmid DNA was isolated using a modification of the alkaline lysis method with SDS [6]. The specific procedure is outlined below.

  • Harvest the cells by centrifugation in a clinical centrifuge (10-min centrifugation. at 2500 × g)

  • Remove the medium by vacuum aspiration or with a Pasteur pipette.

  • Resuspend the pellet in 200 μl of cold alkaline lysis buffer I; transfer the resulting suspension to a microcentrifuge tube.

  • Add 400 μl of alkaline lysis buffer II to the suspension and mix by inverting the tube rapidly about five times (do not vortex). Store the tube on ice.

  • Add 300 μl of ice-cold alkaline lysis buffer III; mix by inverting the tube several times. Allow the mixture to sit on ice for 5 min.

  • Centrifuge at maximum speed for 5 min in a microcentrifuge. Transfer 600 μl of the supernatant to a fresh microcentrifuge tube.

  • Add an equal volume of phenol:chloroform mixture (1:1, v/v) and thoroughly mix the phases by vortexing. Separate the two phases by centrifugation for 2 min at maximum speed in a microcentrifuge.

  • Transfer the aqueous layer to a fresh tube, and add 600 μl of isopropanol at room temperature. Vortex the solution, and allow it to stand at room temperature for 2 min.

  • Centrifuge the mixture at maximum speed for 5 min in a microcentrifuge.

  • Remove the supernatant by vacuum aspiration or with a Pasteur pipette. Invert the tube on a paper towel so that all of the fluid can drain way.

  • Wash the pellet by carefully adding 1 ml of 70% ethanol; centrifuge the mixture for 2 min at maximum speed in a microcentrifuge.

  • Remove the supernatant by vacuum aspiration or with a Pasteur pipette. Remove ethanol from sides of the tube, and allow the pellet to air dry or place in a desiccator until all the moisture has evaporated.

  • Dissolve the pellet in 50 μl of TE (Tris-HCl/EDTA, pH 8.0).

The composition of the lysis buffers used is as follows.

Alkaline lysis buffer I:

  • 50 mM glucose

  • 25 mM Tris-Cl (pH 8.0)

  • Rnase A, 100 μg/ml

  • Lysozyme, 0.5 mg/ml

Alkaline lysis buffer II:

  • 0.2 N NaOH

  • 1% (w/w) SDS

TE (pH 8.0):

  • 10 mM Tris-HCl (pH 8.0)

  • 1 mM EDTA (pH 8.0)

Alkaline lysis buffer III:

  • 5 M potassium acetate, 60 ml

  • Glatial acetic acid, 11.5 ml

  • H2O, 28.5 ml

Each student performed a plasmid preparation, but lab partners pooled samples in preparation for analysis. Following isolation of the plasmid, students were asked to determine the amount and purity of their samples. The first type of analysis used was UV absorption spectroscopy because it is a simple and nondestructive assay. As students have no idea about the amount of DNA they have, they were asked to start with a relatively dilute sample and gradually increase the concentration to obtain an absorbance reading at 260 nm of ∼0.25. They recorded absorbance readings at 260 and 280 nm so that they could quantitate and assess purity.

The next analytical method was fluorometry. The following assay protocol was adapted from the laboratory manual of Sambrook and Russell [6]. A concentrated solution of Hoechst dye was made by dissolving the dye in water at a concentration of 0.2 mg/ml. The working solution was made by adding 50 μl of the concentrated dye solution to 100 ml of fluorometry buffer (0.2 M NaCl, 10 mM EDTA, pH 7.4). Samples contained 3 ml of diluted dye solution and between 5 and 20 μl of DNA sample. Optimal excitation of Hoechst dye occurs at 365 nm, and emission was monitored at 458 nm. Students constructed a standard curve using reference DNA; a range of 50–500 ng was suggested. Based upon the estimation of DNA concentration from UV-visible spectroscopy, they made appropriate dilutions of their samples and took readings at two different dilutions.

The amount of protein in the plasmid samples was determined by using a commercial protein assay that could be adapted to the microscale. Modifications of either the Lowry protocol [7] or the Bradford protocol [8] can be used to quantitate the amount of protein. The reagents for each assay are commercially available from Bio-Rad.

Finally the students analyzed their DNA by gel electrophoresis. They ran a 1% gel in Tris borate/EDTA (TBE) electrophoresis buffer with ethidium bromide (0.5 μg/ml) and the appropriate molecular weight markers. The composition of TBE can be obtained from the laboratory manual of Sambrook and Russell [6].

Allow 2 weeks to complete this laboratory exercise; the isolation and purification was done during the 1st week, and the analyses can be done during the 2nd week. Electrophoresis should be set up during the beginning of the second laboratory period so that it can progress while the other analyses are being performed. Analyses can be performed on a rotating basis so that there is no shortage of equipment.

DISCUSSION TOPICS

Whenever possible students should be given the opportunity to read primary scientific literature; they were asked to read the original paper describing the alkaline lysis method [1]. The paper gives very good descriptions of what happens to the plasmid DNA at each step of the protocol. It was emphasized that most commercially available kits are based upon the alkaline lysis procedure; the major difference is that the plasmid is usually purified using chromatography rather than differential precipitation of genomic and plasmid DNA. To deepen their understanding of the procedure, the students were asked to explain how the pH affects the state of DNA (i.e. why does it denature under strongly alkaline conditions?). They were also asked to explain such things as how SDS interacts with membranes and proteins and how this can lead to precipitation of these macromolecules under high salt conditions. Many students have performed phenol/chloroform extractions on numerous occasions but have never thought about what happens on a molecular level; as part of a written homework assignment, they were asked to explain how and why this extraction functions as a purification step.

Before the students began the spectroscopic analyses, the topics of absorbance and fluorescence were reviewed. Students were reminded that ultraviolet and visible radiation cause excitation of electrons from the ground state to an excited state. Because the absorbance is directly proportional to concentration (Beer's Law), absorbance spectroscopy can be used for quantitative analysis of biologically interesting molecules. This led to a discussion of molar extinction coefficients and the variability with wavelength; both RNA and DNA have maximal absorption at 260 nm, while that of protein occurs at 280 nm. In particular, the molar extinction coefficient of DNA is quite a bit larger than that for protein. Students should conclude that this calls into question the reliability of using the ratio of absorbance at 260 to 280 nm to determine whether there is significant contamination with protein.

Fluorescence spectroscopy is concerned with the radiation emitted as excited electrons relax to the ground state. Fluorescence deals with the transition from an excited singlet state to the ground state; this is best explained to students with the use of energy level diagrams, which can be found in any analytical or physical biochemistry text [9, 10]. The structure of Hoechst 33258 was provided (Fig. 1), and students were asked to hypothesize about how it might bind to DNA; it is known that it does not bind by intercalation [11]. Hoechst dye specifically binds to double-stranded DNA and has minimal interactions with other biological macromolecules. The utility of this dye stems from the fact that its fluorescent yield increases by a factor of 60 upon binding to double-stranded DNA. Base composition of sample, salt concentration, and pH all affect the binding of Hoechst 33258 to DNA. For this reason, assays must be carried out under a standard set of conditions with a reference DNA that has the same base composition as the sample. It was pointed out that fluorometry is a much more sensitive method that UV-visible spectroscopy; fluorometry can be used to detect nanogram quantities of DNA, while accurate quantitation by UV absorption spectroscopy requires concentrations of about 1 μg/ml.

As part of a written homework assignment, students were asked to briefly explain the general principles of electrophoresis. Analysis of experimental data showed that some of the samples had more than one species of plasmid, and this led to a brief discussion of plasmid topology. Also significant RNA contamination will be visible on the gel as a high mobility species with significantly lower fluorescence than plasmid DNA because it is single-stranded and binds less ethidium bromide.

The technical writing assignment for this laboratory exercise was a short report in which there was a brief introduction describing the importance of being able to isolate and purify plasmid DNA and an extensive results and discussion section. There was a range of student data. In a few instances, there was very little discrepancy between the quantitation data obtained from UV absorption spectroscopy and fluorometry. The more common scenario was that data obtained from UV absorption spectroscopy indicated higher amounts of DNA than fluorometry. The goal is for students to explain in the discussion that UV absorption spectroscopy can overestimate the amount of DNA because of interference from other biological macromolecules. There was one instance in which fluorometry gave a higher DNA concentration, and this was most likely due to an error in preparation of the standard curve. Regardless of the data students should be able to support their hypotheses by using the data obtained from protein assays and gel electrophoresis. They should also be able to draw conclusions about the ability of the alkaline lysis method to provide a DNA sample that is free of contaminants. This exercise should also help demonstrate the limitations of each analytical technique.

CONCLUSIONS

This laboratory exercise allowed students to gain practical experience with plasmid preparation and several common analytical techniques used to characterize DNA. One goal of the experience was to encourage students to draw upon knowledge they have gained from the disciplines of biology and chemistry and use it to understand the technical and molecular details of some commonly used procedures in molecular biology/biochemistry. A second goal was to aid students in the development of critical thinking skills in an experimental setting so that they can (a) choose the most appropriate analytical techniques and (b) know the limitations of their data. In fact, several of the students have moved on to research labs and have given positive feedback on the relevance of this particular laboratory experiment to real world settings.

Figure FIGURE 1..

 

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