✦ Theory
Restriction enzymes are endonucleases contained in many bacteria and archea whose function appears to be both maintenance and protection against foreign DNA.
Restriction enzymes function by bonding to the DNA and cutting out (cleaving) damaged portions or, in the case of invading viral DNA, disabling it. Each restriction enzyme has a different recognition site(short sequence) to which it attaches. Recognition sites are short DNA sequences, typically 4 to 8 nucleotides. Below is the recognition sequence of EcoRI2, the restriction enzyme extracted from a strain of Escherichia coli.
5'–GAATTC–3' 3'–CTTAAG–5'
Cells are protected from damage from their own endonucleases by a process known as methylation, in which methyl groups (R–CH3) attach to specific recog - nition sites rendering the DNA an unfit substrate for the enzyme. The following diagram illustrates a typical methylation by the enzyme EcoRI methylase. This methylation prevents attachment by the above-mentioned restriction enzyme EcoRI.
CH3
|
5'–GAATTC–3' 3'–CTTAAG–5'
| CH3
Most recognition sites are palindromes. That is, both strands of DNA have the same sequence of nucleotides when read from the 5' (or 3') end. Each enzyme cuts the DNA at a specific location in the sequence. For example, EcoRI cleaves double stranded DNA as follows:
5'–GAATTC–3' 3'–CTTAAG–5'
Resulting in,
5'–G AATTC–3'
3'–CTTAA G–5'
Many cleavage patterns are possible. In fact, because some enzymes attack multiple variations of a sequence, many more fragment patterns are achievable than the restriction enzymes that perform them. For example, BglI (from Bacillus globigii) recognizes 625 variations of the following sequence:
5'–GCCNNNNNGGC–3' 3'–CGGNNNNNCCG–5'
where N can be A, G, C, or T. The enzyme reads the sequence flanking the variable segment, and will not attach if the nucleotides are not spaced as shown. Table 10-1 lists a few examples of restriction enzymes and cleavage patterns.
Agarose gel electrophoresis3is frequently used in conjunction with restriction enzymes to separate DNA fragments of different sizes. Many variables can affect migration patterns of DNA fragments, relating to shape, size, and a tendency of some fragments to form attach- ments to other molecules. Gen erally speaking, however, small DNA fragments (fewer base pairs) will migrate faster and farther than large fragments (Figure 10-4 and Figure 2 on the Data Sheet). Simply looking at the bands will not tell you the size of the fragments, but running a control sample of known DNA standards in the gel allows construction of a standard curve that can be used to determine unknown fragment sizes.
Because one of the three samples used in this exer- cise is bacteriophage Lambda DNA, special care will be required to achieve a reliable band pattern in the gel.
The phage genome is double-stranded and linear with a 12 base-pair single-stranded tail at the 5' terminus of each strand. These strands are complementary to each other, so when the genome is released inside a host cell the complementary strands link to form a circular mole- cule. In restriction digestion reactions the tendency of
10-2 Restriction Digest 1
1This procedure is adapted, with permission, from Edvotek kit #213 Cleavage of DNA with Restriction Enzymes, for which the company holds copyrights. All materials and equipment required for this exercise can be purchased at Edvotek, Inc., PO Box 341232, Bethesda, MD, 20827, USA. email: info@edvotek.com www.edvotek.com
2Restriction enzymes get their names from a combination of genus, species, strain (when applicable), and a Roman numeral indicating position in order of identification. For example EcoRI comes from Escherichia coli, strain RY13, first restriction enzyme identified from this organism (I).
3This exercise includes agarose gel electrophoresis. Depending on the time available in your lab, your instructor may or may not include gel prepa- ration as part of the exercise. Additionally, because gel preparation, elec- trophoresis, and gel staining are included in more than one exercise, only instructions for running the electrophoresis are included in this proce- dure. A short introduction to gel electrophoresis, agarose gel preparation, and various staining procedures appear in Appendix G.
these cohesive ends or cos sitesto form bonds can reduce the number of fragments desired. Therefore, a short period of heating must be done immediately prior to electro phoresis to minimize this activity.
✦ Application
Restriction enzymes are used extensively in genetic engineering.
✦ In This Exercise
You will be given three samples of DNA and two restric- tion enzymes (EcoRI andBamHI) with which to run a restriction digestion. DNA 1 and 2 are plasmid DNA.
DNA 3 is Lambda phage linear DNA. When your diges- tion is completed, you will run your samples, along with DNA standards (controls), in agarose electrophoresis and use the band migration distance to determine the DNA fragment sizes.
✦ Materials
Per Class
✦one 37°C water bath
✦one 65°C water bath
✦microcentrifuge
EcoRI Escherichia coli 5'–GAATTC–3' 5'–G AATTC–3'
3'–CTTAAG–5' 3'–CTTAA G–5'
TaqI Thermus aquaticus 5'–TCGA–3' 5'–T CGA–3'
3'–AGCT–5' 3'–AGC T–5'
BamHI Bacillus amyloliquefaciens 5'–GGATCC–3' 5'–G GATCC–3'
3'–CCTAGG–5' 3'–CCTAG G–5'
BglII Bacillus globigii 5'–AGATCT–3' 5'–A GATCT–3'
3'–TCTAGA–5' 3'–TCTAG A–5'
HindII Haemophilus influenzae 5'–GTPyPuAC–3' 5'–GTPy PuAC–3'
3'–CAPuPyTG–5' 3'–CAPu PyTG–5'
Sau3A Staphylococcus aureus 5'–GATC–3' 5'– GATC–3'
3'–CTAG–5' 3'–CTAG –5'
SmaI Serratia marcescens 5'–CCCGGG–3' 5'–CCC GGG–3'
3'–GGGCCC–5' 3'–GGG CCC–5'
Pu ⳱Purine (G or A), Py ⳱Pyrimidine (C or T)
TABLE 10-1Restriction Enzymes and Their Recognition Sequences
10-4 RESTRICTIONDIGESTGEL✦This gel contains two lanes from a restriction digest. The bands on the right are from the DNA standard fragments (Markers). The bands on the left are from a DNA sample cleaved by one enzyme. In this photo the wells are at the top and migration was downward.
✦ice bath
✦pipettes and sterile tips to handle volumes from 5 àL to 35 àL
✦five reaction tubes
✦tubes containing:
⽧DNA 1 (on ice)
⽧DNA 2 (on ice)
⽧DNA 3 (on ice)
⽧EcoRI restriction enzyme (on ice)
⽧BamHI restriction enzyme (on ice)
⽧Buffer
⽧10⳯gel loading solution
⽧enzyme grade ultrapure water
Procedure
(Overall time ~ 2 to 4 hours)
Preparation and Digestion Reaction (~ 1 hour: 30 minutes)
Follow the procedural diagram in Figure 10-5.
1 Using the table on the Data Sheet, plan your digestion reactions. (Note:Although you can use any combi- nation of restriction enzymes and DNA samples, it is best not to make it too complicated. Too many small fragments may be difficult to resolve as distinct bands. Large fragments will produce fewer bands, but will contain more biomass and stain more heavily.)
2 Calculate the amount of water required in each reaction tube and enter the data in Table 1 on the data sheet.
3 Obtain all materials including the number of reaction tubes (up to five) you have chosen to use. Place the reaction tubes in a rack along with the DNA stan- dard marker tube. Label the tubes according to your plan entered in the table.
4 Using the plan you set up in the table, add water, buffer, and DNA to the reaction tubes. ALWAYS use a clean pipette for each transfer.
5 Add the appropriate enzyme to each tube and cap tightly. Tap the tubes or vortex them GENTLY to mix the ingredients.
6 Place the tubes in a microcentrifuge for 20 seconds to force the mixture to the bottom of the tube. Be sure to balance the centrifuge before starting the spin cycle.
7 Place the tubes in the 37°C water bath and incubate them for 30 to 60 minutes.
8 When the incubation is complete, remove the tubes and add the 5 àL of 10⳯gel loading solution to each one. Cap and mix gently.
Gel Electrophoresis (20 minutes to 2 hours, depending on gel size and voltage used)
1 Properly orient and place the gel bed in the electro - phoresis chamber. Remember, the DNA placed in the wells will “run toward red”; therefore, the wells must always be positioned on the negative end opposite the positive “red” terminal.
2 Add ~300 mL buffer to completely cover the gel.
3 Prior to loading the gel, place the reaction tubes in the 65°C water bath for two minutes.
4 Allow the tubes to cool for 2 or 3 minutes and then load 35 àL of each sample into its appropriate well.
5 Immediately plug the red and black leads into the electrophoresis apparatus and power supply, turn on the power supply, and set the voltage to the level established by your instructor.
6 Check to see that it is working properly by looking for bubbles rising from the electrodes.
7 When the dye has migrated approximately 4 cen- timeters from the well, turn off the power supply, re- move the cover, disconnect the cables, and go to Appendix G, Module B—Gel Staining.
8 When finished, record your data and follow the in- structions on the data sheet.
References
Edvotek, Inc. 2001. EDVO-Kit #213 Cleavage of DNA with Restriction EnzymesInstruction Booklet. Edvotek, Inc., PO Box 341232, Bethesda, MD, 20827, USA.
Madigan, Michael T. and John M. Martinko. 2006. Chapter 7 in Brock Biology of Microorganisms, 11th Ed. Pearson Prentice Hall. Upper Saddle River, NJ.
Nelson, David L. and Michael M. Cox. 2008. Chapters 8 and 9 in Lehninger: Principles of Biohemistry, 5th Ed. W. H. Freeman and Company, New York, NY.
Roberts, Richard J. January 1980. Restriction and modifications enzymes and their recognition sequences. Nucleic Acids Research, Vol 8 Number 1, p. 197. Oxford University Press. Oxford, UK.
Rx 1 2 3
37°C
Rx 1 DNA
1
DNA 2
DNA 3 Water Buffer Markers
Rx 2
Rx 3 Rx
4
Rx 5 EcoRI BamHI