Exercise #4B - Physical Mapping of Recombinant Plasmid DNA by Digestion with Restriction Endonucleases.

I. Overview:

Physical mapping of restriction endonuclease cleavage sites is generally a preliminary step in the characterization of genes or gene fragments isolated through recombinant DNA technology. In addition, identification of differences in restriction enzyme cleavage patterns between individuals has become an important tool for the diagnosis of many genetic diseases. Restriction enzymes are site-specific endonucleases; that is, they cut DNA molecules only at sites where a specific sequence of bases occurs that the enzyme recognizes. Thus, a restriction enzyme cuts large DNA molecules into a number of fragments with sizes defined by the distribution of the particular recognition sequence. DNA can be digested with several restriction enzymes that cut at different sites and the size of the resulting fragments can be determined by agarose gel electrophoresis. By comparing the sizes of fragments produced by digestion with single enzymes and combinations of enzymes, the cleavage sites can be mapped with respect to each other and a physical map of the initial piece of DNA can be deduced. In this exercise, you will analyze the recombinant plasmid DNA that you prepared during the previous laboratory period. The human DNA insert contained in your unknown recombinant plasmid will be mapped by digestion with the restriction enzymes Eco RI, Hind III, and Bam HI, both singly and in combinations. You will measure the size of the fragments resulting from each digestion and then deduce the order in which the fragments occur.

II. Background:

A. Restriction Endonucleases:

Restriction endonucleases are enzymes that cleave both strands of double stranded DNA after recognizing specific nucleotide sequences on the molecule. Three types of restriction endonucleases have been described. Type I restriction endonucleases recognize a specific sequence but cleave the DNA randomly at some distance away from the recognition site. This type of restriction enzyme requires ATP, Mg++, and S-adenosylmethionine for activity. Type II restriction endonucleases recognize a specific sequence and cleave the DNA at a specific site, usually within the recognition sequence. They require only Mg++ for activity. Type III restriction endonucleases require ATP and Mg++, but cleave DNA 24-26 bases to the 3' side of the recognition sequence.

The principal biological role of restriction endonucleases is thought to be protection against viral infection. Restriction enzymes function in prokaryotic organisms as part of multi-enzyme host restriction and DNA modification systems. Restriction enzymes do not cleave the DNA of the host organism, even though the recognition site (restriction site) may occur many times in the host genome. This is because a DNA modification enzyme, which is usually a methylase with the same recognition sequence as the endonuclease, modifies the potential cleavage sites in the cellular DNA. Since DNA replication is semi-conservative, one strand of newly replicated DNA will contain the modified base(s). Half modified restriction sites are not substrates for the endonuclease. Half modified sites are, however, recognized by the modification enzyme, which rapidly modifies the newly synthesized DNA strand. DNA from an infecting virus that was not replicated in a host cell containing the particular restriction/modification system will not contain modified restriction sites and will be cleaved by the endonuclease. Hence the name "restriction" system. Restriction enzymes "restrict" the host range of bacteriophage to strains of bacteria containing the identical set of restriction/modification genes present in the strain in which the virus was originally replicated.

Because of their specificity, type II restriction enzymes have been important tools for the analysis, mapping, and cloning of DNA. At present, over 400 type II enzymes have been identified from a variety of bacterial strains. These enzymes recognize specific palindromic sequences (usually 4 or 6 base pairs) and cleave DNA at specific sites within the sequence. The asymmetric cleavage of DNA within the palindromic sequence by certain restriction enzymes generates single stranded tails of DNA that may base pair. For example, the enzyme Eco RI recognizes the sequence


 


and cuts the DNA in both stands at sites internal to the recognition sequence. (the arrows indicate the site of cleavage on each strand). The staggered cleavage generates two equimolar molecules with 5' tails.

 



Several different restriction endonucleases such as Bam HI and Hind III generate 5' tails or overhangs. Others such as Pst I, which recognizes the sequence




 


generate tails at the 3 'end. Both 5' and 3' tails are known colloquially as cohesive or "sticky" ends. Some enzymes such as Hae III, which recognizes the sequence

 


 


generate blunt (or flush) ends. Over 100 different sequences are recognized by the over 400 known type II restriction endonucleases. Enzymes that recognize the same sequence are known as isoschizomers. Some isoschizomers cut the recognition sequence in the identical manner while others recognize the same sequence but cleave the DNA at different sites (ie. some give flush ends rather than cohesive ends). Still other isoschizomers recognize the same sequence but may not cleave DNA if it is methylated. The broad spectrum of restriction endonuclease specificities provides a wide range of options for selective cleavage of DNA molecules and for the generation of cohesive ends for intermolecular ligation.

B. Cleavage of DNA with Restriction Endonucleases:


Purified preparations of restriction enzymes are available from commercial suppliers in concentrated stock solutions, which are stable for several months when stored at -20o. Enzyme stocks are stored in buffers containing 50% glycerol. The glycerol acts as an antifreeze and protects the enzymes from the denaturation that would occur if freezing were to take place. Enzyme activity is standardized by the supplier in terms of the ability of a preparation to cleave a reference DNA under a defined set of incubation conditions. One unit of enzyme activity is the minimum amount of enzyme required to completely cleave 1 ug of the reference DNA in 1 hr. The reference DNA (a plasmid or viral DNA preparation) used for different restriction enzymes varies, since the number of cleavage sites in a particular DNA molecule will not be the same for different enzymes. Thus, the amount of enzyme necessary to completely cleave an unknown DNA molecule must be determined by trial. In the laboratory, however, it is usually sufficient to add about 10 times more enzyme units than would be required to digest the reference DNA. This "over digestion" also minimizes the effects of enzyme inhibitors that may be present in crude (rapid lysis) preparations of plasmid DNA.


Restriction enzymes have distinct optima for ionic strength, pH, and Mg++ concentration. Digestion of DNA under conditions that vary too much from the optima results in reduced rates of digestion and sometimes in cleavage at incorrect sites. Since it is not convenient to make up digestion buffers specific to each restriction enzyme, most laboratories use a simple set of 3 or 4 different buffers that vary only in ionic strength. In this laboratory exercise, we will use a single all purpose buffer with the following composition:


100 mM Tris, pH 7.5

50 mM NaCl

10 mM MgCl2

1 mM dithiothreitol

100 ug/ml BSA


This buffer is optimal for digestion by Eco RI and gives about 80% maximum activity for Bam HI and Hind III. The dithiothreitol and BSA reduce enzyme denaturation during digestion. The BSA also tends to bind impurities present in crude DNA preparations (eg. traces of phenol or SDS) that could inhibit the restriction enzyme.


Digestions are usually set up in small volumes (5-20 ul) using 5X or 10X concentrated buffer solutions.


C. DNA Mapping


Several techniques have been used to construct maps of restriction enzyme cleavage sites on DNA molecules. These include single and multiple digestion; secondary digestion with another enzyme after isolating an endonuclease-generated fragment; partial digestion of a 32P end-labeled fragment; and digestion of a DNA molecule with exonuclease for different periods of time followed by digestion with the restriction enzyme of interest. The technique chosen is determined by the level of detail required. Often a combination of procedures must be used to construct a highly detailed restriction map. For construction of a simple map (ie. where only a few cleavage sites are to be determined) analysis of the products of single and multiple enzyme digestions is usually sufficient to deduce the order of the fragments.


In single and multiple enzyme digestion mapping, the restriction sites of one enzyme are oriented with respect to the restriction sites (reference sites) of a "reference" enzyme. The best starting reference enzyme is the one that generates only 2-3 fragments, ie., 2-3 sites on circular DNA or 1-2 sites on linear DNA. Once fixed reference sites are established, a series of double digestions (multiple digestions) can be performed to determine the distance to the restriction sites of the other enzymes. Double digestions are performed either by digesting the DNA molecule with both enzymes simultaneously or sequentially. Fragments that "disappear" or change in mobility after the second digestion contain sites for the second enzyme. New bands appearing represent unique double digestion products. The molecular size of the fragments is determined and, from analysis of all data, a physical map can be deduced. Solving restriction mapping problems is in many ways similar to solving a puzzle. Trial and error is involved in ordering the fragments in different ways until a fragment order is obtained that will produce the observed digestion products.


III. Procedure:


A. Enzyme Digestion:


1. Dilute your sample of unknown recombinant plasmid DNA (from exercise 4A) to give a final concentration of 100 ug/ml. Use TE buffer to make the dilution.


2. Set up 7 reactions on ice according to the table below. Use 500 ul microcentrifuge tubes. You must be very careful in pipetting the correct volumes! Make sure that you know how to use the P-20 Pipetman correctly before you attempt to assemble the reactions. You will be using very small volumes and any significant error will ruin your results!



Reaction:            1          2          3          4          5          6          7

5X buffer          2 ul       2 ul       2 ul       2 ul       2 ul       2 ul       2 ul

DNA                5 ul       5 ul       5 ul       5 ul       5 ul       5 ul       5 ul

H2O                 3 ul       3 ul       3 ul       3 ul       3 ul       3 ul       3 ul

Eco RI             -          1 ul         -         1 ul         -         1 ul        -


Bam HI            1 ul       -            -         1 ul       1 ul         -          -


Hind III             -           -           1 ul        -         1 ul       1 ul        -

 

Be sure to add the restriction enzyme last! Vortex the reaction mix gently.


2. Incubate your reactions in a 37o water bath for one hr.


3. While your samples are digesting, prepare a 1.5% agarose gel using 30 ml of melted agarose. Allow the gel to solidify for at least 0.5 hr. Place the gel in the gel box and cover with running buffer containing 0.5 ug/ml ethidium bromide.


4. After the digestion time is over, remove your samples to an ice bath and add 2.5 ul of Sample Loading Buffer. Vortex to mix.


5. Load your digestions on your agarose gel.


6. Add a lane (#8) containing molecular size markers (Hind III digested lambda phage DNA) and electrophorese at 50V until the tracking dye is within 1 cm of the bottom of the gel.


7. Remove the gel and photograph your results using the UV transilluminator. Be sure that the sample wells are visible in your photograph.

IV. Evaluation of Data:

1. Carefully measure and record the distance that each molecular size marker band has migrated from the original position in the sample well.


2. Measure and record the distance that each of your digestion products has migrated from the original position in the sample well.


3. Plot the mobility of the molecular size standards vs. the log of the molecular size using 3 cycle semi-log paper. Be sure that you understand how to use semi-log paper. Ask your instructor to explain this to you if you are uncertain of how to do this.


4. Determine the molecular size of each digestion product from the plot of your molecular size standards. Again, ask your instructor for help if you are unsure.


5. In a table, indicate the number of restriction products obtained with each digestion and their size in kilobase pairs (kb).


6. Deduce the order and map distance (in kb) of the restriction sites found in your recombinant plasmid. Draw a restriction map of the plasmid.


Answer the following in the Discussion section of your lab report:


1. Are any partial digestion products visible on your gel? How large are they and which uncleaved restriction sites do they contain? (If they occur, these can be very useful in deducing the order of the fragments, since they contain fragments that are located next to each other.)


2. Are any bands present in the undigested plasmid (reaction #7) not cleaved by the restriction enzymes? What might these bands be?

 

References:

Dillon, J-A. R., Nasimm A., and Nestmann, E. R. (1985) Recombinant DNA Methodology, John Wiley & Sons, Inc., New York, pp 35-40.


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