An introduction to restriction mapping of DNA

48

An Introduction to Restriction Mapping of DNA C E HEPFER and S L TURCHI

Departments of Biology and Chemistry Millersville University of Pennsylvania Millersville, PA 17551, USA Introduction Restriction enzyme mapping is a powerful tool for the analysis of DNA. This technique relies on restriction endonucleases, hundreds of which are now available, each one recognizing and reproducibly cleaving a specific base pair (bp) sequence in double-stranded DNA thus generating fragments of varying sizes. Since the rate at which a D N A molecule moves through an agarose gel during electrophoresis is inversely proportional to its size, 1 the lengths of these D N A fragments can be ascertained and this information used to determine the positions of cleavage sites in a DNA molecule. Comparative analysis of fragments generated by cleavage with two different restriction endonucleases enables the molecular biologist to determine the relative location of particular recognition sequences in the DNA molecule. Restriction mapping has widely publicized applications. These include DNA fingerprinting, the detection of genetic defects and the sequencing of the human genome. Although students readily appreciate the importance of this new methodology, they often find it difficult to understand the techniques and logic involved. The experiments and mapping exercises described below introduce students to the methods used in the restriction enzyme analysis of DNA. This laboratory has been used effectively in both introductory and advanced undergraduate courses. The use of predigested DNA fragments helps to insure the success of the experiment and to reduce costs. Restriction mapping exercises are designed to provide students with tangible experience in the abstract reasoning required for accurate interpretation of results. These exercises are meant to be carried out during electrophoresis of student samples.

Aim of the Practical (1) To demonstrate laboratory techniques of agarose gel electrophoresis and restriction endonuclease digestion of DNA which are used in restriction mapping. (2) To provide practice in applying abstract reasoning required in comparative restriction analysis of DNA.

buffer as described by Maniatis et al. 2 Three application wells will be needed for each student or group. One well per gel should be reserved for the molecular weight standard.

Supplies Each student or group will need 3 sterile microcentrifuge tubes, one P20 Pipetman (or comparable microsampling devices), 10 sterile pipette tips, and plastic gloves. Each laboratory will need a water bath with microcentrifuge rack at 65°C, ice bath, Vortex mixer, microcentrifuge, power supplies (50 volts), plastic trays for staining, UV light source (300 nm) and bleach.

Experimental Procedure

Sample Preparation Label three sterile microcentrifuge tubes 1, 2 and 3. Into tube No 1, place 8 p~l of EcoRI-digested lambda DNA, into tube No 2 place 8 p.l of HindIII-digested lambda DNA, and into tube No 3 place 8 p.l of lambda DNA digested with both of these restriction enzymes. Heat the tubes for 5 min in a 65°C water bath. This insures separation by preventing fragments from adhering to each other. 3 Quickly cool each tube in the ice-bath. To each of the three tubes, add 2 p.l of tracking dye, mix by vortexing, and centrifuge for 1 min in a microcentrifuge.

Electrophoresis Working from left to right, place 10 ~1 from tubes 1, 2 then 3 into adjacent sample wells in the agarose gel. Each well should contain the contents of only one tube. A 10 p~l sample of the molecular weight standard should be applied to one well on each gel. Carefully add running buffer to each of the buffer chambers so that it covers the edges of the gel without flowing over the top. Electrophorese samples at 50 volts until the tracking dye approaches the end of the gel (approx 90 min). (While the gel is running, restriction enzyme mapping exercises 1, 2 and 3 may be performed.) When the tracking dye has moved sufficiently far, carefully remove the gel with its underlying glass plate from the unit. The instructor then stains the gel in ethidium bromide for 15 min. Ethidium bromide (a cancer causing agent) attaches to the DNA and allows it to be visualized, as fluorescent bands, under ultraviolet light. The gel is removed from the stain, rinsed with water and placed on a black background for visualization with ultraviolet light. Wearing gloves, mark the location of bands observed in each lane with a common pin. Compare the results obtained with those outlined in restriction mapping exercise 3.

Reagents In advance of the laboratory, prepare 1000 ml TPE

Questions Did you see the same number of fragments? Are the relative sizes of fragments, as estimated by the distance moved, similar to the values given in the restriction mapping exercise?

Running Buffer, 300 ml ethidium bromide staining solution, and 10 ml gel loading buffer (type III) as directed by Maniatis et al. 2 Caution: ethidium bromide, a powerful mutagen, should be handled with care. It may be inactivated with household bleach. Store solutions at 4°C and warm to room temperature before using. Lambda DNA solutions: obtain one vial (0.25 units) each of EcoRI digest, HindIII digest, EcoRI and HindIII digest (Sigma Chemical Co). To each vial, add 200 ~1 of sterile running buffer. Allow several hours for the D N A to go into solution. Dispense 50 ~1 of each into sterile microcentrifuge tubes and store up to 48 h at 4°C. Molecular weight standard: to 16 p.1 of the HindIII-digested Lambda DNA solution add 4 p.l of tracking dye. Apply 10 ~1 to one sample well. Eight fragment bands (23 130 bp, 9416 bp, 6557 bp, 4361 bp, 2322 bp, 2027 bp, 564 bp, and 125 bp) should be visible after staining. Agarose gels (0.8% w/v): immediately before the laboratory, prepare gels in small horizontal electrophoresis units using TPE

The results reported in the following exercises (Figures 1-3) were obtained from experiments similar to the one carried out by the students. Three distinct molecules of DNA were digested with either EcoRI, HindIII or both enzymes. EcoRI recognizes the bp sequence G A A T T C / C T T A A G and cuts both DNA backbones within this site. HindIII recognizes and cleaves within a different bp sequence (AAGCTY/TTCGAA). The sizes of the DNA fragments generated by each type of digestion were determined by agarose gel electrophoresis in comparison to molecular weight standards. In each of the exercises, the goal is to determine the location of all EcoRI and HindIII cleavage sites along the DNA molecule. The positions of some of the fragments generated are already indicated on the restriction maps. It is necessary to determine the position of all remaining fragments. Start by carefully cutting out the fragments generated. Use a straight edge to compare the positions of cleavage sites on lines 2, 3 and 4

Materials

B I O C H E M I C A L E D U C A T I O N 17(1) 1989

Restriction mapping exercises

49 Restriction Map

1

100

3 4

Fragments Generated Hind III (line 3)

b.\\\\\~,~,-~\\\~ I ~ \ \ \ \ \ \ \ \ \ \ \ ~ \ \ \ \ \ \ \ \ \ \ ~

(Figure 1). Now it should be possible to place the HindlII fragments correctly on line 3 (Fig 1).

Exercise 2 Starting at the right side of the restriction map (Figure 2), determine which HindlII fragment is located at this end. Notice that a 15 bp double-digest fragment (line 4) lies adjacent to the 40 bp EcoRI fragment located at this end of the DNA (lines 2 and 4). Since 15 + 40 = 55, it can be concluded that the 55 bp HindlII fragment must be located at the right end of this D N A molecule. Place this fragment in position on line 3.

EcoRI & Hind III (line 4)

Restriction Map

1

Figure 1 Exercise 1: Diagrammatic representation of the fragments generated when a 100 base pair (bp) molecule of DNA (line 1 of the restriction map) is cleaved with EcoRl (~7] line 2), HindlI1 ( [ ~ line 3), or doubly-digested with both of these restriction enzymes ( [ ] line 4). Fragments-generated which are not shown on lines 2-4 are represented in appropriate scale below the restriction map. Numbers on fragments indicate size in bp

(Figures 1-3). If a fragment on line 2 (EcoRI) overlaps a fragment on line 3 (HindIII), the size of the-overlapping region will correspond to a double-digest fragment. The size of a single digest fragment will be equal to the sum of the sizes of all doubledigest fragments contained within (overlapping) this larger fragment. Since double-digest fragments result from cleavage with both EcoRI and HindIII, the position of cleavage sites on line 4 of each restriction map must correspond to the location of cleavage sites on either line 2 or line 3. The guidelines provided for each exercise should assist in placing fragments correctly on the restriction maps.

Note Enlarging Figures 1-3 helps students more easily to manipulate the fragments generated. Exercise 1 When the 100 bp D N A molecule shown in Fig 1 is cleaved with EcoRI, two fragments are generated. Since the fragments are 20 bp and 80 bp in size, these results indicate that the sequence G A A T F C / C T T A A G is located at a position 20 bp from one end of the D N A molecule. The restriction map (Fig 1, line 2) shows that this 20 bp EcoRI fragment is located at the left of the 100 bp D N A molecule. Cleavage of another copy of the same 100 bp D N A molecule with HindIII yields two fragments, 35 and 65 bps in length. Does this give any information about the location of the AAGCTT/ T F C G A A sequence in this D N A molecule? Obviously the HindIII cleavage site is located 35 bp from one end of the D N A molecule, but which end? Is it at the same end as the EcoRI site? Attempt to place the HindIII fragments in the correct position on line 3 of the restriction map (Figure 1). Double digestion with both restriction enzymes may provide information that will help. When the same 100 bp D N A molecule is cleaved with both EcoRI and HindIII, three fragments, 35, 20 and 45 bps in length, are generated (Fig 1). The 35 bp end fragment generated by HindIII alone is still intact, but the 65 bp fragment has been cut into smaller pieces (45 bp and 20 bp) by EcoRI. The 20 bp double-digest fragment corresponds to the same-sized fragment generated by EcoRI alone. Since the 20 bp EcoRI fragment is contained within the 65 bp HindIII fragment, the positions of these two fragments must overlap. The 80 bp fragment generated by EcoRI alone has been cleaved into two fragments (35 bp and 45 bp) by HindlII. Does this give any information about the position of the 35 bp HindlII fragment relative to the 80 bp EcoRI fragment? Position the double-digest fragments on line 4 of the restriction map

BIOCHEMICAL EDUCATION 17(1) 1989

/

soo

l

4a2 V.A~./;//////'~fd///////2"/////f.~////~ Fragments Generated Hind III (line 3)

EcoRI & Hind III (line 4)

Figure 2 Exercise 2: Diagrammatic representation of the fragments generated when a 500 base pair (bp) molecule of DNA (line 1 of the restriction map) is cleaved with EcoRl ( [ ] line 2), HindlII ( [ ] line 3), or doubly-digested with both of these restriction enzymes (~t~ line 4). Fragments-generated which are not shown on lines 2-4 are represented in appropriate scale below the restriction map. Numbers on fragments indicate size in bp A comparison of fragments on lines 2 and 4 indicates that the 160 bp EcoRI fragment is cut into pieces by HindlII. One of these is the 15 bp fragment that is already in position on line 4. Since 160 - 15 = 145 (the size of an existing double-digest fragment), it can be concluded that the 145 bp double-digest fragment must lie adjacent to the 15 bp fragment. Correctly position the 145 bp fragment on line 4. This 145 bp fragment must be contained within one of the HindlII fragments. Only the 195 and 170 bp fragments are large enough to contain a fragment of this size. To decide between these two possibilities, subtract 145 bp from the length of each of these two fragments. Since 195 - 145 = 50 and no 50 bp double digest fragment exists, it can be concluded that the 145 bp fragment is contained within the 170 bp HindlII fragment (170 145 = 25) and that the 25 bp double-digest fragment lies adjacent to the 145 bp fragment. Position these fragments on lines 3 and 4. Using the same type of logic, continue to determine the positions for the remaining D N A fragments.

Exercise 3 Start at the right side of the restriction map (Fig 3). Which HindlII fragment is at this end? It is known that the 3500 EcoRI fragment is adjacent to a 800 bp double-digest fragment. Knowing that 3500 + 800 = 4300, deduce the size of the HindlII fragment which overlaps these two fragments. Comparison of lines 2 and 4 indicates that the 800 bp doubledigest fragment is contained within a 6000 bp EcoRI fragment. Which double-digest fragment must lie immediately to the left of this 800 bp fragment? Could it be the 4000 bp fragment (800 + 4000 = 4800)? What about the 5200 bp fragment? Continue to place fragments until all cleavage sites are located. Remember that the locations of cleavage sites on line 4 must correspond to those on either line 2 or line 3.

50 Restriction Map 1 2 4

.7

.8

Fragments Generated

Hind III (line 3)

utions, blood, packed cells, plasma or albumin on circulatory and renal function can be determined. This is interesting and useful, and it is a great pity that the program is so cumbersome. The presentation is old fashioned and one feels that there must be a more attractive way of presenting the results. The program is certainly worth a try, but I suspect many might be turned off by the on-screen presentation. J F B Morrison

EcoRI & Hind III (line 4)

Book Reviews Figure 3 Exercise 3: Diagrammatic representation of the fragments generated when a 28 000 base pair (bp) molecule of DNA (line 1 of the restriction map) is cleaved with EcoRI (1[~ line 2), HindllI ( [ ] line 3), or doubly-digested with both of these restriction enzymes ( [ ] line 4). Fragments-generated which are not shown on lines 2-4 are represented in appropriate scale below the restriction map. Numbers on fragments indicate size in kilobase pairs (kbp)

References 1Alexander, R R, Griffiths, J M and Wilkinson, M L (1985) Basic Biochemical Methods, John Wiley and Sons, New York 2Maniatis, T, Fritsch, E F and Sambrook, J (1982) MolecularCloning. A Laboratory Manual, Cold Spring Harbor Laboratory 3Sigma Chemical Company 1988 Catalog

Microcomputer Software Reviews MacDope and MacPee m IRL Press Medical and Physiological Simulations IRL Press Ltd, PO Box 1, Eynsham, Oxford OX8 1JJ, UK. 1987. Both for I B M - P C / X T / A T with graphics adaptor. Require 512K. Macdope: £125 + V A T or $250, MacPee: £275 + V A T or $550. [See also Biochemical E d u c a t i o n 16, 108 (1988) for reviews of companion programs MacPuf and MacMan: combined price for all packages £395/$790. Demonstration discs available.] MacDope models the pharmacokinetics of many common modem drugs, and combines this with the ability to change the physiological handing of these drugs, by altering the volume of distribution and the half-times of excretion and metabolism. This program may be useful for students of medicine, physiology, pharmacology and biochemistry. It teaches how to write prescriptions, the pharmacokinetics of common drugs and the characteristics of patients that influence the efficacy of drug action. As with other members of this family of programs, I found the presentation somewhat tedious, but the dedicated student will undoubtedly get a grasp of pharmacokinetics by persevering with the program. MacPee is described as a simulation of heart, peripheral circulation, kidneys, body fluids, electrolytes and hormones. The program gives students the opportunity to perform experiments on a model patient that would otherwise be impossible or dangerous. For instance, the effects of parenteral or oral isotonic, hypotonic or hypertonic saline, dextrose, KCI sol-

BIOCHEMICAL EDUCATION 17(1) 1989

Clinical Studies in Medical Biochemistry Edited by R H Glew and S P Peters. pp 259. O x f o r d University Press, M a d i s o n A v e n u e , N e w York. 1987. $35 or $18.95 I S B N 0 - 1 9 - 5 0 3 9 2 1 - 1 or 0 - 1 9 - 5 0 3 9 2 2 - X With the pace of biochemical research being as rapid as it is today, the major and perennial task of convincing preclinical medical students of the central and crucial role of basic biochemistry in the understanding of human disease, its diagnosis and therapy should be becoming even easier. However, the problem often facing the (often) nonclinical teacher of preclinical biochemistry is one of accessing, assimilating and summarizing all the various facets of a particular clinical problem in a form suitable for presentation, be it in lecture or seminar format. 'Clinical Studies in Medical Biochemistry' goes a long way towards this goal. In assembling the contributions of a large number of experts in their fields, the avowed aim of the editors (R H Glew and S P Peters, University of Pittsburgh Medical School) was to provide 25 self-contained clinical problems covering a very wide range of biochemical interest, each of which is presented in sufficient depth to form the basis of a single lecture or seminar if required. The standard format consists of case history and physical examination, biochemical investigations with normal controls, diagnosis and therapy, with a central section on the biochemical perspectives including relevant pathways, etc. References, mostly to reviews, and follow-up questions are included. Cases presented include many of the 'standard' metabolic disorders - glucose 6-phosphate dehydrogenase deficiency, pyruvate dehydrogenase deficiency/congenital lactic acidosis, PKU, errors of urea cycle, sphingolipidosis (Gaucher's), lysosomal disorders (I cell disease), diabetes and sickle cell anaemia. Others cover more wide-ranging ground, not usually seen in such books, such as bacterial toxins (diphtheria, cholera), hyaline membrane disease, anaphylaxis, angiotensin and hypertension, familial hypercholesterolaemia, etc. The arrangement is claimed to parallel typical medical biochemical courses, though I found groupings to be rather curious (the editors admit other scenarios are possible). However, the value of this volume lies in its use as an extension of standard biochemical texts, a valuable resource of illustrative and challenging material to back up almost any component of a preclinical course. Excellent though this book will be for teachers, I do not see it as a required text for preclinical courses in British medical schools. The depth of coverage is probably somewhat greater than the student will require. Encourage your students to consult it from time to time, precise selected examples for your own teaching, and use it for setting challenging examination questions! S J Higgins