Introduction
The discovery of restriction enzymes (REs), bacterial transformation, and agarose gel electrophoresis caused the sudden increase of popularity of molecular biology in the 1970s. Restriction enzymes allow the manipulation of specific sites of an organism’s genes and are commonly used in many laboratories worldwide. Restriction enzymes cut DNA in many different ways and can produce sticky ends and blunt ends (Weaver, 2012). The most abundant type of restriction enzymes is Type II, which require magnesium cations as cofactor (Pingoud et al., 2005). Aside from their popularity is recombinant DNA technology as a “cut and paste” tool, estriction enzymes can also be used to create maps of plasmids, which are circular extrachromosomal DNA commonly found in prokaryotes (Reece, 2004).
RE-digested DNA is usually separated according to size through agarose gel electrophoresis (AGE) (Reece, 2004). Electric current guides the DNA fragments through a sort of maze of agarose polymers and the fragments get separated according to size. In this experiment, plasmid DNA is digested by two restriction enzymes: NcoI and SalI in both single and double RE digestion reactions. The digested DNA is analyzed through agarose gel electrophoresis to create plasmid maps.
Methods
Restriction Enzyme (RE) Digestion
Three separate reactions were prepared for digestion with NcoI, SalI and both NcoI & SalI; these were carried out in 10µL reaction volumes. The following components of the RE digestion were added chronologically and on ice: DEPC water (6.5µL or 6µL), 10X NEB Buffer 3 (1µL), 10X BSA (1µL), DNA (1µL) and enzyme (0.5µL each). The solution was homogenized by gentle pipetting and was placed in a 37°C water bath for an hour.
Agarose Gel Electrophoresis (AGE)
One percent (1%) w/v agarose gel was prepared by dissolving agarose in 1X Tris-Acetate-EDTA (TAE) buffer and heating in a microwave. Once cooled to the touch, 50 mL of the agarose solution was poured into the casting tray. The comb was then placed and the gel was left to cure for 20 to 30 minutes. The solidified gel was then transferred into an electrophoresis apparatus with the wells nearer to the cathode. The chamber was then filled with 1X TAE buffer.
Two and a half (2.5) µL of 5X loading buffer was added to the digestion reactions. Ten (10) µL of the samples were loaded into the wells of the gel. Finally, 5µL of the DNA ladder HyperLadder™ 1kb (Bioline) was loaded into the last well. The apparatus was run at 110V for 35 to 40 minutes. The gel was viewed using a blue-light transilluminator to see the SYBR Green-stained DNA. The gel was duly documented and was then disposed into the appropriate waste container.
Results
Figure 1. Agarose gel electrophoresis of RE-digested plasmid DNA. Lane 1: digested with NcoI; Lane 2: digested with SalI; Lane 3: digested with NcoI and SalI; M: Molecular weight marker (HyperLadder™ 1kb)
Figure 2. Log10 size (base pairs) and migration distance of individual bands of the molecular weight marker plotted in a graph. Trendline analysis gave an equation y= -0.3374x + 4.6585 and linearity of 0.9763 (Microsoft Excel 2010).
Data Analysis
The individual bands on the agarose gel were measured out arbitrarily from the top of the wells using the provided image. The bands of the molecular weight marker HyperLadder™ 1kb have known base pair sizes and were used as standard for the calculation of the sizes of the digested plasmid DNA. Figure 2 is a graph of the log10 size of the molecular weight ladder bands and their migration distances. Using the equation obtained from trendline analysis, y= -0.3374x + 4.6585, and since that y = log10BP, a formula for calculating the sizes of DNA bands in the agarose gel can be derived:
BP=10(-0.3374x+4.6585)
where BP is the base pair size of the band and x is the migration distance of the band in the gel. A sample calculation for the bands of the single RE digestion, which had a similar migration distance:
BP=10(-0.3374(2.4)+4.6585)
BP=7059
Finally, the calculated sizes of the bands of the RE digestion are shown in Table 1.
Discussion
Restriction Enzymes
Restriction enzymes, also known as restriction endonucleases, are proteins that recognize specific base sequences in double-stranded DNA and cleave these sequences at specific sites in both strands of the duplex (Berg et al., 2002; Weaver, 2012). They were first discovered in E. coli were discoved in the late 1960s by Stewart Linn and Werner Arber. They are found in many prokaryotes, wherein their biological role is to cleave foreign DNA molecules (Berg et al., 2002; Gilbert, 2000). The restriction sites within the cell's own DNA is not degraded because they are methylated (contain methyl groups) (Pingoud et al., 2005). Most sites are palindromic, or in other words, are inverted repeats (Bickle and Krüger, 1993; Weaver, 2012). They are useful in manipulation of genes and other molecular biology techniques.
Restriction enzymes can have the same restriction site with other enzymes. They are called isoschizomers if they cut the same recognition sequence in the same way. Otherwise they are called heteroschizomers (Reece, 2004). Table 2 shows a number of restriction enzymes. SalI and RflFI, EcoRI and FunII, and PstI and BspMAI are all isoschizomer pairs. Cloning can be done by treating the plasmid and the insert using different RE’s that are isoschizomers. This is especially useful when the insert and the plasmid don’t have overlapping restriction sites.
Agarose Gel Electrophoresis
In electrophoresis, DNA is separated as they migrate from a negative- to positive- charged anode, since the nucleic acid backbone is negative. The agarose gel acts like a sieve which limits the movement of the DNA based on its size. Because of this, the heavier or longer DNA will move slower and would stop at an earlier level, while lighter or shorter DNA would migrate faster and stop farther in the gel. It is a simple and highly effective method for separating, identifying and purifying 0.5- to 25-kb DNA fragments (Ausubel, 2003; Sambrook and Russell, 2001).
AGE typically involves three general steps. First is the preparation of a gel with an agarose concentration sufficient for the required resolution. Agarose concentrations of 0.3 to 2% are most effective to resolve nucleic acids. The general rule is that the higher the concentration, the better resolution of small fragments. The second step is sample loading and running at a voltage and time for optimum separation. Lower voltage and longer time is usually done for better resolution of the bands. The third and final step is staining either by in-gel staining or post-staining and visualization of the gel by illumination with certain lights. Ethidium bromide is usually used to stain nucleic acids but it is highly toxic and carcinogenic. Safer dyes can be used such as GelRed or SYBR Green (Ausubel, 2003; Sambrook and Russell, 2001; Boyer, 2000).
The mobility of nucleic acid in agarose gels is also affected by the molecular conformation of the nucleic acid. The most common DNA conformations are superhelical circular (form I), nicked circular (form II) and linear (form III) (Boyer, 2000). This implies that a molecular weight marker (linear DNA) standard can only be used to calculate or estimate the size of bands of linear DNA. That is, the size of native or undigested plasmids cannot be reliably estimated because it is usually supercoiled. Since a supercoiled plasmid is more compact, it migrates faster than its linear counterpart.
The applications of agarose gel electrophoresis include identifying DNA fragments based on size as well as purification of desired DNA fragments through gel excision. Using a molecular weight marker which contains DNA fragments of known sizes as a reference, the size of a DNA fragment may be approximated. Molecular weight markers are usually packaged at known DNA concentrations and this allows the semi-quantification of the DNA concentration of the sample using the relative intensities of the bands. This is particularly useful for plasmid ligation reactions wherein the plasmid to insert ratio must be optimal for ligation to occur. Finally, AGE also allows for purification of desired DNA fragments through the use of several gel extraction kits wherein the fragment is excised from the gel and spun down in a spin column to remove the agarose debris. This is particularly useful for separating DNA fragments with similar but resolvable lengths using AGE (Sambrook & Russel, 2001).
In Figure 1, the 200-bp band of HyperLadder™ 1kb ladder was not resolved. This could mean that there was too little amount of the DNA to be resolved. Another possible explanation for this is that the smallest band could have run off the gel, although it is very unlikely because the apparatus was not run for too long. There also seems to be contamination between lanes 2 and 3. A small band about 5kb in length can be seen in lane 2. This could have been caused by improper loading of the sample in the well. From Table 1, the size of the plasmid is estimated to be 7059 bp through single RE digestion and 7696 bp through double RE digestion. The double digestion seems to be a more reliable estimate because the fragments were more linearized. The single digestion reactions show wavy and “tailed” bands that could indicate that the plasmid is still coiled to a great extent and could have migrated faster in the gel.
Plasmid Reconstitution
AGE analysis of the NcoI, SalI double digestion of the plasmid revealed two fragments approximately 5.8 kb and 1.8kb in length (see Figure 1). Reconstitution analysis suggests two possible configurations or locations of the restriction sites that would yield the aforementioned fragments when the plasmid is digested by both enzymes.
Figure 3. Possible restriction enzyme cut sites in the plasmid. Data from the double digestion was used to reconstitute these two possible restriction sites in the plasmid.
The uncertainty of the positions of the cut sites can be solved by the addition another enzyme that only has one restriction site within the plasmid, such as FatI. Double digestion with using three enzymes would yield three unique combinations: FatI & NcoI, NcoI & SalI, and SalI & FatI. There would different fragment lengths per combination that would become puzzle pieces of the plasmid. Analysis of fragment lengths would then reveal one arrangement of these restriction sites, unlike when using only two restriction enzymes.
In conclusion, the experiment was overall successful in the digestion of plasmid DNA using restriction enzymes and in the AGE analysis of digested DNA. The bands were clear enough to be distinguished from one another, albeit one band of the molecular weight marker was unaccounted for. There also seemed to be contamination during sample loading but is easily avoidable through practice. A broad plasmid map was also successfully elucidated through analysis of fragment lengths. A recommendation for future experiments would be to use another enzyme in order to successfully determine the location of restriction sites within the plasmid.
References
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Gilbert, H.F. (2000). Basic Concepts in Biochemistry (2nd Ed.). New York: McGraw-Hill, Inc.
Pingoud, A., Fuxreiter, M., Pingoud, V. and Wende, W. (2005). Type II restriction endonucleases: structure and mechanism. Cellular and Molecular Life Sciences 62: 685-707.
Reece, R.J. (2004). Analysis of Genes and Genomes. England: John Wiley & Sons, Ltd.
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Weaver, R. (2012). Molecular Biology (5th Ed.). New York: Mc-Graw Hill, Inc.
