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Restriction enzymes (RE), also called restriction endonucleases are specific enzymes that protect bacteria against foreign DNA. The mechanism of work is cutting the single or double-stranded DNA at specific recognized terminals called restriction sites, generating 5'-phospahte and 3' OH ends. Restriction enzymes are divided to three major categories which are (Types I, II and III). Basically, this classification is based on the composition of their subunit, specific position for cleavage, sequence arrangement, and cofactor requirement. The first two categories of restriction enzymes, type I and III enzymes have multiple subunits which improve their restriction and modification activities. The modification activity of these restriction enzymes makes them resistant to prevent degradation of the host's DNA. Chemically, this process is happened by addition of methyl (CH3-) groups to nucleotides through the recognition sites. These types of enzymes can cut the DNA chain randomly, not exactly at their recognition sites. Similar to most of the biological transformations these enzymes also use ATP as source of energy for their translocation. However, the main drawback which incorporated with these enzymes is that they do not produce districted fragments which can follow by restriction sites. Another type of restriction enzymes are type III. The major difference for this group compared to type I and type II of enzymes is the source of energy, since they do not use ATP hydrolysis for nucleolytic activity. They are able to discern palindromic sequences in couple of base pairs and this recognition make them efficient for DNA cleavage using Mg2+ through their recognition sites (Micklos, et. al 2003,Aggarwal, et. al 1995; Pingoud, et. al 2001;). There are different applications of restriction enzymes digestion including: DNA fingerprinting, molecular cloning, polymorphism studies (SNP identification), genomic analysis and restriction mapping. DNA fingerprinting is the method that scientists used to understand that are different samples related to the same source (person or animal). They compare a small part of sequences of DNA which is not similar among individuals. By analyzing those sequences they can say what the probability of a match is. Molecular cloning is the method for isolation of a specific DNA sequence and then proliferate that sequence in vitro condition to achieve multi copies of that. Another application of restriction enzyme digestion is single nucleotide polymorphisms (SNP). SNP are DNA sequence difference which is the result of alteration of a single nucleotide (A,T,C,or G) in the genome sequence. About 99% of human DNA sequence are similar, and difference in DNA sequence have a main role in human resistance to disorders, bacterial infections, viruses, toxins, and etc. This key feature makes SNPs precious forbiomedical investigations and improvement of medical treatments. Agarose Gel Electrophoresis is a useful tool for the separation of DNA fragments based on their rate of movement through an electric field that is proportional to their molecular sizes.
The purpose of this experiment is to use restriction digestion techniques to identify two plasmids containing either HCE1 or HCE 2 and also identify of the type II restriction enzyme used based on the digestion patterns.
Enzyme digestion was performed using 15 uL of plasmid, 1 uL of enzyme, and 15 uL of Buffer 2 which including: (NaCl (50mM), Tris-HCl (10 mM), MgCl2 ( 10 mM), Dithiothreitol (1Â mMÂ )) (New England Biolabs). The mixture was gently mixed and then incubated in at 37o C in water bath for 1 hour. After incubation, the digest process was stained with ethidium bromide. Agarose gel electrophoresis was employed to analyze. The gel was run for 1 hour and visualized using Typhoon 9410 Variable Mode Imager (Amersham Biosciences, NJ). Data analysis was performed using Typhoon Scanner 3.0.
Results and Discussion
Two unlabelled plasmids containing either HCE1 or HCE 2 were digested using different enzymes including: BamH I, Hind III, Pst I or Sac I for 1 hour at 37 oC. The digested fragments were separated using agarose gel electrophoresis. Various groups of combination of buffer solution, DNA samples, and enzymes are presented in Table1. 1kb plus DNA ladder (Invitrogen) as standard marker was loaded on electrophoresis gel.
Restriction Enzyme (G)
Table1. Data for used material that for each lane are presented in Table1, including: each lane contains which buffer, restriction enzyme, and plasmid.
Substrates were digested by enzymes in an appropriate buffersolution including NaCl (50mM), Tris-HCl (10 mM), MgCl2 (10 mM),andÂ Dithiothreitol (1Â mM) for 45 min at 37oC. Samples were stained in ethidium bromide and analyzed by 0.8% agarose gel at 90V. Lane 1: Invitogen 1 Kb plus DNA ladder, Lane2-9: student samples Figure1.
Figure1. Restriction digestion pattern, two unknown substrates were digested by an unknown enzyme in a suitable buffer (NaCl (50mM), Tris-HCl (10 mM),MgCl2 (10 mM),Â Dithiothreitol (1Â mM)) for 45 min at 37oC. Samples were stained in ethidium bromide and analyzed by 0.8% agarose gel at 90V. Lane 1: Invitogen 1 Kb plus DNA ladder, Lane2-9: student samples.
Fragments were obtained through electrophoresis and the sizes for fragments were in the range of 300bp to 6500 bp. As it is shown in Figure 1, in sample number 3, HCE plasmid was digested into 3 different fragments in the size of 450 bp, 1350 bp and 5000 bp. These experimentally obtained sizes of fragments were compared to the known HCE2 restriction enzyme pattern (Yan, 2010 and presented in Table 2) and showed the same pattern with HCE2 digested by Pst I. The same procedure was used to interpret the data for each sample and find the enzyme and buffer. Therefore, based on the theoretical restriction sites the expected fragment lengths were calculated (Table 2.Fragment sizes were estimated using the 1kb plus DNA ladder (Invitrogen) Table 2.
HCE1 (6425 bp)
HCE2 (6221 bp)
BamH I (6)
Hind III (4)
Pst I (2)
Sac I (8)
BamH I (7)
Hind III (5)
Pst I (3)
Sac I (9)
650, 1100, 4900
320, 1400, 4800
1800, 1 5100,
450, 1350, 5000
1900, 5000, 6200
Table2. The data obtained by student containing restriction sites, cleavage positions, employed enzyme for each lane, and fragment size. 1. Red numbers suffers from star activity and they can be digested incompletely.
Table2 contains information about: determined identities of the lanes, student experimental fragment size which achieved by student, Restriction sites, and cleavage sites. Obtained experimental data by student compared to expected data to understand which enzymes used for each sample (lane). As it is shown in Figure1 and Table2, digestion of HCE1 using Pst I generated three fragments around 323bp, 1370bp, and 4732 bp and digestion of HCE2 using Pst I generated 3 fragments size around 438 bp, 1350 bp, and 4693 bp. Following the same pattern for other samples, it was obtained that digestion of HCE1 using Hind III produces three fragments with the size of 650bp, 1100bp, and 4900 bp and digestion of HCE2 using Hind III generated one fragment with the size of 6220 bp. This single band showed this sample could be either digested incompletely, or only one restriction site worked to cut the DNA sample into a single fragment.
Two fragments were obtained from digestion of HCE1 using BamH I in the range size of 1700bp and 5050bp. The same restriction enzyme used for HCE2 and two fragments in the sizes of 900bp and 5000bp were observed. In the case of digestion of HCE1 by Sac I five fragments were obtained with the size of 550bp, 1400bp, 1800bp, 5100bp, and 6200bp. In this case some of the fragments were appeared to be loaded not carefully or suffered from incomplete digestion. These fragments are showed in red color in Table 2. Also, digestion of HCE2 using Sac I enzyme three fragments were obtained in the size range of 1900bp, 5000bp, and 6200 bp. In order to obtain a satisfactory efficiency from restriction enzymes the experimental conditions should be optimized. These variables in conditions include: the temperature of incubation, the composition of buffer, ionic strength, and corresponding pH. Another source of the error is the human error during the loading gel for electrophoresis. These types of errors were observed in this experiment (Figure1, Lanes 5, 8, and 9). Difference in the intensity of bands represents the quantity of the fragments compared to each other. The band intensity is related to two main parameters such as: high sample concentration and loading error. If the experimental conditions do not meet the optimized requirements, incomplete digestion or "star" activity would be observed. Star activity is the incident in which the enzymes cut different sites from the canonical cleavage sites by single base substitutions. As it was observed there was a small deviation in the fragments size from the expected sizes which can be the consequences of mutations and/or impurities in the DNA sequence. Because of mentioned problems during the work, it is highly recommended to use more information and an improved the skill of researchers during the experiment.