Restriction Enzyme Concentration Directly Related to Digestion Progress

AbstractRestriction enzymes are found in bacteria and function to help protect the bacterial genome from attack by viruses. However, the enzymes have profound impacts in biotechnology as well. Enzyme kinetics explains that increasing the concentration of enzyme should accelerate the reaction progress, until the reaction has completed. In this experiment, restriction enzyme HindIII was added to bacteriophage DNA in various concentrations, and the progress of the digestion was measured with an agarose gel. After comparison to the DNA ladder, it was determined that a restriction enzyme concentration of at least 6%, or 2 microliters per microgram of DNA, is needed for complete digestion to occur. Knowing this value for various enzymes can lead to the most effective choice of an enzyme for a given reaction. Therefore, this research has implications on increasing the efficiency of biotechnological restriction enzyme applications, such as RFLP digests, which can help identify genotypes of people prone to certain diseases.IntroductionRestriction enzymes are found in bacteria and protect the cell from invasion by foreign bodies. The enzymes cut the foreign DNA at various sites, to prevent their incorporation into the genome and subsequent replication. Type two restriction enzymes are endonucleases (cut inside the double stranded DNA) and can cut at different places depending on the nature of the restriction site. Restriction enzymes recognize a specific site on the DNA, which is typically a certain palindromic sequence of nucleotides. The palindrome is significant because it allows the enzyme to cut both strands of the DNA, since the sequence is the same forwards and backwards. Different restriction enzymes have been identified in different bacteria, and in addition to their natural functions, have biotechnological applications. For example, restriction enzymes have been used to insert genes coding for human protein into bacteria, to get increased expression of important proteins. One recent example of cloning with restriction enzymes is with alkaline protease genes from extremophiles. These genes are of interest because they can withstand extreme conditions. The researchers cut the genes out of the organisms with restriction enzymes in E. Coli, and were able to efficiently and inexpensively clone the gene of interest (1). In this experiment, the restriction enzyme HindIII was used. HindIII is a common restriction enzyme with a known cut site, and has been used for identification of certain polymorphisms. Restriction fragment length polymorphisms (RFLPs) are analyses that look at differences in genes that happen to be located in restriction enzyme sites (2). Through RFLP analysis, it has been shown that HindIII can help identify the genes responsible for hemophilia (3) and genes with implications in hypertension and type 2 diabetes (4). If RFLP analysis is inexpensive, then such screenings could be routine and help prevent the onset of such diseases. The progress of a restriction enzyme digest is measured on an agarose gel. The gel is made of a porous semisolid material that allows smaller molecules to travel through it faster. A current is applied (positive at the top and negative at the bottom) to the gel container and the negatively charged DNA molecules travel through towards the positive end. The smaller molecules travel further and the larger molecules get stuck closer to the top. The size of the DNA bands on the gel can be determined by comparison to the DNA ladder. Knowing the size of the bands can be used to determine the location of the restriction enzyme sites on the molecule of interest. It can also be used to determine if the digestion has gone to completion, because a complete digestion will have many short bands and an incomplete digestion will have more large bands and will not have as many small bands.In order to efficiently complete a restriction enzyme digest, the enzymes must be at certain conditions that allow maximal activity. These factors can include temperature, pH, buffer type or concentration, and restriction enzyme concentration. For this experiment, the effect of restriction enzyme concentration on the digestion process was examined. Enzymatic kinetics states that enzyme concentration does affect the rate of the reaction, because each enzyme can act upon a finite number of substrates per unit of time (5). After a certain point, increasing the concentration of substrate while holding the enzyme concentration constant will have no effect on the reaction rate because the enzyme is saturated (5). However, adding more enzymes will change the reaction rate because now there are more active sites available for catalyzing the reaction (5). Additionally, adding more enzyme while keeping the substrate concentration constant will also exhibit an eventual leveling off because the substrate will eventually run out. Each restriction enzyme has an experimentally determined measure of activity known as the unit. The unit is defined as the amount of enzyme needed to completely digest 1 microgram of DNA in 1 hour at 37 degrees Celsius (6). This experiment will determine the unit for HindIII. It is hypothesized that increasing the restriction enzyme concentration will result in more digested DNA molecules, as visualized on the gel with bright short bands, up to a certain point at which the DNA is completely digested and adding more enzyme will have no effect.TubeDNA (microliters)10X Buffer (microliters)Restriction enzyme (microliters)Water (microliters)

1110.08.0

2110.27.8

3110.47.6

4110.67.4

5110.87.2

6111.07.0

Table 1: Reaction components for each tubeThe experiment is conducted with six tubes of varying restriction enzyme concentrations. The reaction ingredients for each tube are shown in Table 1 below. The six tubes are incubated for one hour at 37 Celsius. The negative control for the experiment is the tube with 0 microliters of DNA, because there is no reaction occurring. The positive control is the tube with 1 microliter of DNA, because it is the point at which the restriction enzyme constitutes 10% of the reaction mixture, the maximum it can be while still working properly (6). Following the incubation, the digestion mixtures are run on a 1% agarose gel. ResultsThe gel was run at 113 volts for 45 minutes. The gel was visualized by adding ethydium bromide, which intercalates with the DNA, making it appear bright when shined under UV fluorescence. The gel was photographed as shown in Figure 1. The distances traveled for the bands in each lane are presented in Table 2. The tubes with 0.2 and 0.4 microliters of restriction enzyme show incomplete digestion, and the tubes with 0.6, 0.8, and 1.0 microliters of restriction enzyme digestion show complete digestion as defined by comparison to the positive control. Therefore, the restriction enzyme concentration must be at least 6% for the reaction to go to completion and the unit Figure 1: Photograph of garose gel of different reaction digestions Lane 1: None Lane 2: Tube 1 Lane 3: None Lane 4: Tube 2 Lane 5: None Lane 6: Tube 3 Lane 7: Tube 4 Lane 8: Tube 5 Lane 9: Tube 6 Lane 10: 10 microliter DNA ladder Lane 11: 5 microliter DNA ladder Lane 12: Nonefor this restriction enzyme is (0.6 microliters enzyme / 0.3 micrograms/microliter DNA * 1microliter DNA)), 2 microliters of Table 2: Presence of DNA bands as compared to a complete digestion. The bands of the positive control (tube 6) are the reference and whether or not that band is present in each of the other tubes is indicated. Y=band is present and as bright as control; N=band is absent or is present but is not as bright as control.enzyme per microgram of DNA.

TubeBand 1 (0 cm)Band 2 (0.3 cm)Band 3 (0.5 cm)Band 4 (1 cm)Band 5 (1.5 cm)Band 6 (1.7 cm)

1YNNNNN

2YYYNNN

3YYYNNN

4YYYYYY

5YYYYYY

6YYYYYY

DiscussionThe restriction enzyme concentration needed for the reaction to go to completion is 6% of the reaction volume. This indicates that for every 1 microliter of DNA, 0.6 microliters of restriction enzyme HindIII is required to fully digest the DNA in one hour. More generally, 2 microliters of HindIII are required for digesting 1 microgram of DNA. Any concentrations less than this do not completely digest the DNA and leave some larger bands, indicating that the DNA has not been cut at all possible restriction enzyme sites. The reaction with no restriction enzyme present has only one band at a bit over 1000 base pairs, indicating this is the size of the uncut DNA. The other DNA molecules have this band as well, indicating that the DNA is not 100% digested, even at the maximum concentration of restriction enzyme. Because this band is present in all samples, including the positive control, for the purpose of this experiment the DNA is still considered completely digested, relative to the positive control. Perhaps a future experiment can use a smaller quantity of DNA, such as 0.5 microliters, and then the band will disappear because the enzyme will cut the DNA completely. However, this experiment presents limitations, such as not having enough DNA to visualize the bands, because they will not be as intense.Another potential source of error for this experiment is using a restriction enzyme that has no restriction sites on the DNA used. If this is the case, there would only be one band in all lanes, regardless of how much restriction enzyme is present. The band would be at the level of band one, indicating that all the DNA remained uncut, because there were no sites to cut at. However, because restriction sites are usually small, it is highly probable that a given strand of DNA has a certain 4-8 nucleotide sequence. The presence of restriction sites can be confirmed by cDNA libraries, genomic sequencing, or by running digests and comparing the lengths of bands present to that of uncut DNA. This was not a concern for this particular experiment, as indicated by bands other than just the band from the uncut DNA.Knowing the restriction enzyme concentration necessary for full digestion provides valuable insight as to the kinetics of the restriction enzyme. Since adding more DNA beyond the 0.6 microliters does not create a visible change in the number or intensity of bands, the enzyme is likely operating at its full potential at this point and is digesting DNA as much as it can. However, this does not explain the saturation point of the enzyme. Rather, it shows that the enzyme has a point at which the concentration of enzyme does not affect how much DNA can be cut. This experiment begs two more questionsat what point is the enzyme saturated with DNA and does adding more enzyme quicken the reaction completion time when compared to that without enzyme. In order to test the saturation point of the restriction enzyme, the enzyme concentration can be held constant and the DNA concentration can be changed. The concentration at which increasing the amount of uncut DNA does not change the digestion bands is the saturation point for the enzyme. Another experiment can be conducted to determine if adding more enzyme changes the time it takes to complete the reaction. A set concentration of DNA will be used and the amount of enzyme and the time monitored will be changed. This experiment would require multiple assays and gels, as it is more complicated. The first gel can have a small enzyme concentration and each lane would be stopped at different times. The second gel would have a larger enzyme concentration and the lanes would be stopped at the same times as those of the first gel. Theoretically, increasing the enzyme concentration increases the rate of reaction, thereby decreasing the amount of time necessary for digesting one sample of DNA. Therefore, the reaction with the highest enzyme concentration should have the least amount of time required for full digestion, as visualized by seeing full digestion in lanes with the reaction stopped at earlier stages when compared to those with less enzyme concentration. This experiment sets the groundwork for developing these other future experiments, and has implications for biotechnology as well. For example, since varying restriction enzyme concentration affects the digestion completion, biotechnology companies can be more efficient and cost-effective with their experiments. If a researcher wanted to determine if the HindIII polymorphism responsible for hemophilia (4) or for hypertension and type 2 diabetes was present in a large, impoverished population, the approach should be inexpensive (5). Using the minimum amount of restriction enzyme required to fully digest the sample would be the most cost-effective method, and therefore the values determined in this experiment can help improve the efficiency of such large screenings. References(1) Gohel S.D., and Singh S.P. (2012). Cloning and expression of alkaline protease genes from two salt-tolerant alkaliphilic actinomycetes in E. coli. Int J Biol Macromol. 50:664-671.

(2) Cox, M.M., Doudna J., ODonnell M. Molecular Biology (1st edition). W. H. Freeman, 2011.

(5) Tymoczko J., Berg J., Stryer L. Biochemistry: A Short Course (2nd edition). W. H. Freeman, 2011.

(6) Kadandale, P. Molecular Biology Lab M116L. 2013.

(3) Dubey AK, Hussain N, Mittal N. (2010). HindIII-based restriction fragment length polymorphism in hemophilic and nonhemophilic patients. J Nat Sci Biol Med. 1:25-8.

(4) Munoz-Barrios S., Guzman-Guzman I.P., Munoz-Valle J.F., Salgado-Bernabe A.B., Salgado-Goytia L., Parra-Rojas I. (2012). Association of the HindIII and S447X polymorphisms in LPL gene with hypertension and type 2 diabetes in Mexican families. Dis Markers. 33:313-20.