restriction enzyme mapping of a bacteriophage lambda dna

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Refe rences

biochemical tool. The technology involved in producing them at present is expensive and time- consuming and the objectives involved should be weighed carefully before embarking on monoclonal antibody production. Scaling production up to industrial levels can also prove difficult and costly. At the present time, monoclonal preparations used in cancer patients have been of murine origin, but their use over extended periods may introduce the possibility of adverse allergic reactions similar to serum sickness. Human hybridomas are being developed, 22 but progress is slow. There can be little doubt that in the future, such antibodies will play a major role in the diagnosis and treatment of human disease.

1 Kb'hler G and Milstein C (1975) Nature 256,495-497 2 Harris H and Watkins J F (1965)Nature 205,640-646

3 Harris H (1970) 'Cell Fusion'. Clarendon Press, Oxford 4 0 i V T and Herzenberg L A (1980) in 'Selected Methods in Cellular Immunology'. Edited by Mishell B B

and Shiigi S M, W H Freeman, San Francisco, 351-372 5 Fazekas de St Groth S and Scheidegger D (1980)JImmunol Methods 35, 1-21

6 Zola H and Brooks D (1982) in 'Monoclonal Hybridoma Antibodies'. Edited by Hurrell J G R, CRC Press Florida, 1-57

7 Kb'hler G, Howe S C and Milstein C (1976) Fur J Immunol 6, 292-295

8 Schulman M, Wilde C D and Kb'hler G (1978)Nature 276, 269-270 9 Kearney J S, Radbruch A, Liesegang B and Rajewsky K (1979)JImmunol 123, 1548-1550

lO Littlefield J W (1964) Science 145,709-710

11 Dulbecco R and Freeman G (1959) Virology 8, 396 12 Moore G E, Gerner R and Franklin H (1967) JAMA 199, 519-524

la Iscove N N and Melchers F (1978) JExp Med 147, 923-933 14 Isaacs A and Lindenmann J (1957) Proc Roy Soc, Series B 147, 258-267 15 Secher D S and Burke D C (1980)Nature 285,446-450 16 Webster R G, Kendal A P and Gerhard W (1979) Virology 96,256-264 17 Gerhard W, Yewdell J W, Frankel M E and Webster R G (1981) Nature 290, 713-717 18 Wiktor T J, Flamand A and Koprowski H (1980) J VirolMethods l, 33-46 19 Tedder R S, Yao J L and Anderson M J (1982) JHyg 88, 335-350 2o Yoshida N, Nussenzweiz R S, Potocnjak P, Nussenzweiz V and Aikawa M (1980) Science 207, 71-73 21 Olsnes S (1981) Nature 290, 84 22 Sikora K, Alderson T, Philips J and Watson J V (1982) Lancet i, 11-14

Introduction

RESTRICTION ENZYME MAPPING OF BACTERIOPHAGE LAMBDA DNA

NOREEN CUNNINGHAM, JAMES TOMLINSON and FRANCES JURNAK

D e p a r t m e n t o f Biochemis t ry Universi ty o f California Riverside, California, USA

News coverage of potential medical applications and fmancial success stories in the field of genetic engineering have captivated students' curiosity and aroused hopes of future employment pros- pects. To the research scientist, genetic engineering is a potentially powerful technique which may be used to solve fundamental biological problems but is not necessarily worthy of the intense, almost fanatical, student interest. Unless the faculty is populated with practicing genetic engineers, the topic does pose a dilemma for an undergraduate biochemistry laboratory curri- culum. How does one capitalize upon the intense student interest in the topic with either little expertise or minimal financial support for an undergraduate laboratory?

Several years ago, we introduced a restriction enzyme mapping experiment into the inter- mediate biochemistry laboratory course in which 50-60 juniors and seniors are enrolled. Al- though the experiment is not truly representative of many DNA recombinant research problems, it does introduce the student to the basic biochemicals and techniques used frequently in genetic engineering. Not only does the experiment elicit a favorable response from the students, but the final analysis of the data is challenging to all. From the instructor's viewpoint, the experiment requires minimal preliminary preparation and is surprisingly inexpensive to conduct. Most of the specialized equipment required can be adapted from electrophoresis apparatus already present in a student laboratory.

Biochemical Education 11(4) 1983

T h e E x p e r i m e n t

131

Bacteriophage lambda DNA is incubated with different Class II restriction enzymes and the cleavage products are analyzed by agarose gel electrophoresis. The objective of the experiment is to determine the number and size of the DNA restriction fragments and to map the relative order of all restriction sites on the lambda DNA.

Phage lambda DNA is a double-stranded, linear molecule, 49130 base pairs in length. 1 Class II restriction enzymes recognize and cleave double-stranded DNA at specific base pair sequences; 2 therefore, cleavage of the lambda DNA by a Class II restriction enzyme generates a unique set of nucleic acid fragments? The digestion products are separated by agarose gel electrophoresis. The negatively charged nucleic acid fragments, which have a constant charge/mass ratio, migrate in an electric field within the agarose medium as a function of size. From the number of fragments, the number of restriction sites on the lambda DNA can be deduced. Also, the unknown molecular weight of each DNA fragment can be determined by comparing the electrophoretic mobility of the unknown fragment to that of a standard set of DNA fragments of known size. Cleavage of lambda DNA with Hind III generates a reliable set of standard fragments whose known sizes span a broad molecular weight range.

In the experiment, lambda DNA is incubated with a set of three unknown restriction enzymes, X, Y and Z, and with dual combinations of all three, XY, YZ and XZ. The fragments generated by each restriction cut are sized on an agarose gel by comparison to the known Hind III fragments. The fragmentation information for one restriction enzyme is arbitrarily chosen to establish the relative location of a set of cleavage sites which will serve as landmarks along the DNA. The cleavage sites for the second and third restriction enzymes will be mapped relative to the first set. If only the digestion experiments with a single restriction enzyme are considered, it quickly becomes apparent that there are many possible arrangements of the restriction sites which are consistent with the data. To determine the correct arrangement, the fragmentation patterns from DNA digestion by all dual combinations of the restriction enzymes, XY, XZ, YZ (double-digest) must be analyzed. A simplified version of the strategy is illustrated in Figure 1. Consider a linear DNA sequence that is 1000 base pairs in length. Restriction enzyme X cleaves the DNA at one site, generating two fragments of size 200 and 800 base pairs. As depicted in Figure 1 A, the fragmentation pattern of X is used arbitrarily to assign the relative left-right orien- tation. Digestion of the DNA by restriction enzyme Y also results in two fragments of size 350 and 650 base pairs. Relative to the restriction site of X, there are two possible arrangements of the Y cleavage as shown in Figure lB. The ambiguity is resolved by consideration of the results of the digestion of the DNA with both X and Y. The DNA is cut twice, generating fragments of 150, 200 and 650 base pairs. Only one of the alternative arrangements is compatible with the fragmentation results of the double digest.

a . X digest 200 ~ 800

b. Y digest

350 ~ 650

650 ~ 350

Figure I Lambda DNA digests

200

c. XY alternatives

2OO

,50 ~ 6~

450 ~ 350

For the experiment, only one set of three unknown restriction enzymes is required. However, the particular set of restriction enzymes can be varied from class to class to discourage students from seeking the correct mapping answer from previous classes. Therefore, we have included


132

Materials

Exper imen ta l P ro toco l

herein a protocol description for three different sets of unknown restriction enzymes. To simplify the final digestion pattern, each set has been carefully chosen so that two of the three restriction enzymes cleave lambda DNA at only one or two locations. We have found that Set 1 (Bam HI, Xba I, Kpn I), Set 2 (Sma I, Xho I, Xba I) or Set 3 (Kpn I, Xba I, Xho I) are three sets which yield unambiguous results. If financial resources are severely constrained, the experiment can be modified to include digestion by only the first two restriction enzymes listed in each set.

[Note The concentration of various reaction components, including salt, glycerol and restriction enzyme, is known to alter the DNA sequence specificity of the restriction enzyme reaction. The preliminary preparation and Final experimental conditions have been carefully chosen to optimize the restriction enzyme activity and specificity.]

Restriction enzymes, lambda DNA, electrophoresis grade agarose and ethidium bromide were purchased from Bethesda Research Laboratories (BRL), Maryland, USA. Each restriction enzyme was obtained as a lyophozyme, a form which is stable indefinitely at --20°C. The restriction enzymes were reconstituted with the accompanying dilution buffers according to the BRL instruc- tions to a final concentration of 4 units//A for Sma I and 8 units/ttl for all remaining restriction enzymes. Although restriction enzymes are available from different biochemical companies, BRL was selected as the supplier for reasons of restriction enzyme cost, stability and reliability. For classes with more than 50 students, it is more economical to purchase the restriction enzymes as a BRL Maxi Set II which includes 2000 units each of six restriction enzymes and 500 #g of lambda DNA. Unused restriction enzymes in the lyophozyme form may be stored indefinitely for future use. Without any discount, the cost of the required enzymes and DNA for 27 groups is approximately $320 or $12 per group. If financial resources are a strong consideration, the concentration of the lambda DNA and the restriction enzymes may be reduced by 50% but the lower concentrations may affect the cleavage specificity of the restriction enzymes.

For the cleavage reaction, each restriction enzyme requires a specific buffer, given in ten- fold stock concentrations as follows: Buffer H (Hind III) 200 mM Tris HC1, pH 8.0, 70 mM MgC12,600 mM NaC1; Buffer S (Sma I) 150 mM Tris HC1, pH 8.0, 60 mM MgC12, 150 mM NaC1; Buffer X (Xba I) 60 mM Tris HC1, pH 8.0, 70 mM MgC12, 1 M NaCI; Buffer Y (Xho I) 80 mM Tris HC1, pH 7.4, 60 mM MgCI2, 60 mM 2-mercaptoethanol, 1.5 M NaC1; Buffer B (Barn HI) 200 mM Tris HC1, pH 8.0, 70 mM MgC12, 1 M NaC1 and Buffer K (Kpn I) 60 mM Tris HC1, pH 7.4, 60 mM MgC12, 60 mM 2-mercaptoethanol, 60 mM NaC1. The cleavage reaction is quenched by the 'Stop Buffer' containing I00 mM EDTA, 2% bromphenol blue, 1% SDS and 50% glycerol. A 1% agarose solution (w/v) is prepared by melting agarose at 80°C in Electrophoresis Buffer (40 mM Tris acetate, pH 7.8, 20 mM sodium acetate, 2 mM EDTA). The final agarose gels are stained in 2.5 mg/liter ethidium bromide (a possible carcinogen) and destained in water.

Restriction Enzyme Reaction The components of the restriction enzyme reaction are added to a reaction vessel in a specific order. For cleavage with one restriction enzyme, combine 37/al sterile water, 5 /21 of 10 × restriction enzyme buffer, 3/21 restriction enzyme and 5/al lambda DNA (0.3 mg/ml). For cleavage with two restriction enzymes, combine 34 #1 sterile water, 5/A of 10 × restriction enzyme buffer, 3/al of each restriction enzyme and 5 #1 of lambda DNA. In the cleavage reaction by two restriction enzymes, choose the reaction enzyme buffer with the higher ionic strength. Prepare a sample of uncleaved DNA for the gel by diluting 5/21 of lambda DNA with 45/al of sterile water. Swirl the tubes gently and incubate at 37°C. After one hour, add 5/21 Stop Buffer to each reaction vessel and heat to 65°C for 5 minutes to prevent reanneal- ing of the restriction fragments. For comparative purposes, the entire sample should be loaded onto the agarose gel in the following order: (1) Set 1 : Hind III, lambda DNA, Xba I, Xba I-Kpn I, Kpn I, Barn HI-Kpn I, Bam HI, Barn HI-Xba I, (2) Set 2: Hind III, Sma I, Sma l.Xba I, lambda DNA, Xba I, Xba I-Xho I, Xho I, Sma I-Xho I or (3) Set 3: Hind III, lambda DNA, Xba I, Kpn I- Xba I, Kpn I, Kpn I-Xho I, Xho I, Xho I-Xba I. Agarose Gel Electrophoresis Horizontal agarose slab gels are prepared in a simple, noncommercial apparatus, the design of which may be modified to accommodate available laboratory supplies. The apparatus consists primarily of a five-sided rectangular plexiglass box, measuring 15 cm in width, 30 cm in length and 6 cm in depth. Two plexiglass strips (15 cm × 0.5 cm × 4 cm) are inserted across the 15 cm width of the box and sealed to the bottom of the box, approximately 7 cm from each end. The inserted strips create an inner 4 cm high support for the agarose gel plate and two well separated reservoirs at each end of the box. Platinum wire electrodes are connected to a banana plug adaptor and inserted into each reservoir.


133

Results

In preparation of the agarose gel, 1 cm cubes of plexiglass are placed at the four corners of a glass plate (16 cm × 15 cm). Each cube is attached by a small piece of tape extending from the cube to the underside of the plate. To form a raised edge on the plate, two cubes are connected with tape, of which a 0.5 cm edge is firmly pressed against the underside of the plate to prevent leakage. All cubes are connected in a similar fashion to form an appropriate cast for the agarose gel.

A 1% agarose solution is preheated to 60-80°C and gently but quickly poured over the surface of the glass plate to a depth of 3 - 4 mm. The plate is gently tilted back and forth to insure that the liquid agarose covers the entire surface and then positioned on the two support strips in the plexiglass box on a fiat surface. A ten-weU comb is carefully inserted into the gel perpen- dicular to the plate to a depth of 1 mm less than the thickness of the gel. The comb must be clamped in place. After 20 minutes, the gel comb is carefully removed. The agarose is allowed to solidify for an additional 30 minutes in the horizontal position. Then the glass plate is carefully lifted from the apparatus and the tape is removed from the edges. The agarose gel is repositioned in the apparatus and the samples are loaded into the best eight wells, omitting the edge lanes if possible. The electrophoresis buffer is added to both reservoirs and the power supply is connected. Electrophoresis is carried out for 5 - 6 hours at a constant voltage of 100 volts. To avoid a bowed appearance of the dye in the gel, electrophoresis is carried out in a cold room. When the dye front has migrated to the edge of the gel, the gel is removed, stained in ethidium bromide solution for 30 minutes and destained in water for 15 minutes. The gel is placed on a UV light box and photographed for one second with a Polaroid MP-4 lens at f 5.6 using an orange filter and Polaroid film, Type 57. If photographic equipment is not available, the gels may be sealed in plastic wrap and the measurements made directly on the UV light box. Safety glasses must be worn at all times to prevent UV damage to the eyes. Note Due to the large pore size in the gel matrix, agarose gels are prone to breakage and must be handled very carefully throughout the preparation and final analysis. The gel matrix can be strengthened by increasing the agarose concentration to 1.3%, but the larger molecular weight fragments in the experiment will not clearly be resolved in the smaller pore size. Although a 0.7% agarose gel gives very good resolution, the average student is able to carry out the electrophoresis more successfully with a 1% agarose gel which separates the high molecular weight fragments adequately for the final interpretation.

Agarose gel analysis The migration rate of a macromolecule in an applied electric field is proportional to the charge of the macromolecule and inversely proportional to its mass and shape. Be- cause nucleic acids have an evenly distributed, negatively-charged phosphate backbone, the charge/mass ratio is constant for all sizes and does not significantly affect the migration rate. The electrophoretic mobility of DNA fragments is primarily dependent upon the shape of the molecule. All small DNA fragments are rod-like in shape. As the DNA fragment increases in size, the nucleic acid strand folds up and deviates from a linear, rod-like shape in a random manner. The migration rate is also a function of the medium in which the macromolecule travels. In a semi- solid support medium, such as agarose, the pores of the gel matrix function as a molecular sieve, retarding the movement of the larger molecules for a longer time. In the electrophoresis of small DNA fragments on agarose gels, the molecular seive effect predominates and the migration rate varies linearly with the inverse of the logarithm of the molecular weight of the fragment. As the DNA fragment increases in size, the migration rate deviates from linearity as the shape deviates from a rod-like structure.

Although the precise mathematical relationship between mobility and molecular weight is not a simple function, it can be determined empirically by the use of an appropriate set of nucleic acid fragments of known size, such as the Hind III restriction cleavage product of lambda DNA. To generate the standard plot, the migration distance of each Hind III fragment and that of the uncleaved lambda DNA is measured from the top of the agarose gel to the front of each band to within an accuracy of 0.1 mm. The migration distance is plotted on the abscissa as a function of the logarithm of the molecular weight on the ordinate. It is important to obtain accurate measurements and to use a large graph size. Small errors in the migration distance will result in large errors in the molecular weight determination. The plot will be linear only in the lower molecular weight range of the Hind III fragments. Depending upon the exact electrophoretic conditions, two small Hind III fragments of 1965 and 2260 base pairs respectively, should be visible at the lower end of the agarose gel. The smallest Hind III fragments of 98 and 590 base pairs will migrate off the gel under the conditions of the experiment.


134

The migration distance for each restriction fragment generated by each lambda DNA digest is measured and the molecular weight is determined from the standard plot. All fragments from the suggested set of restriction enzymes will fall within the Hind III molecular weight range. In the upper molecular weight range, large changes in the size will result in small differences in the migration distance. Therefore, it is important to determine carefully if the upper bands are singlets or doublets and if the migration distances differ from lanlbda DNA or DNA fragments in adjacent gel lanes. The suggested sample loading sequence was designed to optimize the compari- sons of fragments of similar molecular weight.

The molecular weight of the DNA fragments generated by each restriction enzyme should sum to the molecular weight of tambda DNA, 33.0 -+ 3.3 X 106 daltons. Cumulative errors in electrophoretic migration, the measurement of the migration distances and the interpolation of the standard plot result in a student experimental error of 10-20%. If the sum of the fragment molecular weights is less than 90% of lambda DNA, the fragmentation pattern of the single and double restriction digests must be carefully analyzed to determine if each band represents one or more fragments of similar molecular weight. The suggested sets of restriction enzymes have been chosen so that the student can resolve all such ambiguities by careful analysis of the double digest fragmentation patterns.

After the molecular weight of all restriction fragments have been assigned, the cleavage sites of two restriction enzymes should be mapped relative to those of a designated restriction enzyme following the strategy outlined in Figure 1. If the set of restriction enzymes, Kpn 1, Xho I, Xba I, is used, the fragmentation pattern is simple and the location of all restriction sites is straight- forward. If a set with Barn HI or Sma I is used, additional information is required in order to map the relative location of all cleavage sites. Both Barn HI and Sma I have multiple cleavage sites on lambda DNA; the other two restriction enzymes in each set cleave lambda DNA at only one location. It is, therefore, not possible to deduce the relative location of those Barn HI or Sma I fragments which are not cleaved by the other two restriction enzymes in the double digest experiments. To circumvent the problem without choosing three restriction enzymes that generate a very complicated fragmentation pattern, the student can be given an idealized restriction map for Barn HI or Sma I. The experimental data should be compared to the idealized map to determine the accuracy of the data. The cleavage sites of the two unknown restriction enzymes can be mapped relative to the idealized location of the Barn HI or Sma I restriction sites. Idealized maps for each restriction enzyme are given in Figure 2.

0 100 Lambda

DNA

479 568 765 777 9~2 65.3 824

.r..,, I J 1522 ..'" 1 1 5 I I

113 468 581 713 862 510

I t I I J I t I 1 I

Figure 2

S t u d y questions

References

360 395 69.0

I ii I x"°' I i The cleavage sites on lambda DNA 4 for each restriction enzyme are mapped on a relative scale o f 0 to 100, where 100 represents 33.0 X 10 ~ daltons or 100% o f the lambdagenome.

(a) Design a simple experiment to determine if the restriction enzymes are contaminated with other endo- or exonucleases. (b) Give one reason why restriction enzyme digestion of DNA can yield more information about the structure of DNA than a protease digestion of a protein can ever yield about protein structure. (c) Design an experiment using restriction enzymes to determine if the initial double-stranded DNA sample is linear or circular.

1 D L Daniels, J R de Wet and F R Blattner, J Virology 33, 390 (1980) 2 H O Smith, Science 205, 455-462 (1979) a R J Roberts, CRCCriticalReviews in Biochemistry 4, 123-164 (1976) 4 Product Catalogue, Bethesda Research Laboratory, PO Box 577, Gaithersburg, Maryland 20760, p 160 (1982)

Biochemica l E d u c a t i o n 11(4) 1983

restriction enzyme mapping of a bacteriophage lambda dna

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