Restriction Enzyme Digestions and Plasmid Mapping AbstractPlasmids are engineered as cloning vectors in recombinant DNA technology as it contains important nucleotide sequences needed for DNA cloning.  The aim of this experiment is to determine whether the unknown plasmid DNA is cut into different sized fragments by using a variety of restriction enzymes- namely EcoR1, Pst1 and BamH1. A 1%agarose gel was used to run the digestions and after staining it with ethidium bromide solution, the cleaved DNA fragments were visible under a trans-illuminator using an Ultraviolet light source. The size of the fragments was estimated by plotting a semilogarithmic graph of the distance travelled by each fragment (mm) to the fragment size (in kilobase pairs). The results obtained were used to create two equally plausible plasmid maps to show the sites where the restriction enzymes cleaved the DNA.Introduction Cloning vectors are generally used in recombinant DNA molecules to clone DNA. They act as carriers by carrying foreign DNA sequence into a host cell. This is usually done by inserting DNA fragments of few base pairs into vector DNA. The resultant recombinant DNA is then introduced into the host cell and undergoes replication to form large number of recombinant DNA molecules containing the vector DNA. The most commonly used cloning vectors include E. coli plasmids and bacteriophage lambda vectors.Plasmids are small, circular, double-stranded, self-replicating DNA molecules, also known as extrachromosal DNAs, found naturally occurring in a parasitic or symbiotic relationship in yeast and bacteria. One of the earliest plasmid vectors constructed was pBR322, which contained two different antibiotic resistance genes. Generally, E. coli plasmids are engineered as cloning vectors in recombinant DNA technology as there are shorter in length of approximate 3kb and contain important nucleotide sequences needed for DNA cloning- ‘a replication origin, a drug-resistance gene and an area for insertion of DNA fragments. (ncbi) Plasmids are not only used to clone a DNA fragment to sufficiently amplify it but to also incorporate a foreign DNA into a host cell.To clone DNA, restriction enzymes (bacterial enzymes or restriction endonucleases) are used to cut DNA at specific sites called restriction sites to form small DNA restriction fragments with sticky ends, usually of 4-8bp in length. This, when introduced with into plasmid vector, cut by the enzyme at a single site, bonds with the complementary “sticky ends” of the DNA fragments through phosphodiester bonds. (ncbi) This is followed by gel electrophoresis- a technique used to separate our DNA fragments according to their size against an electric charge. DNA fragments usually move towards the positive end of the electrophoretic tank as they are negatively charged, which is pH dependant. Based on their size and charge, smaller fragments move faster and quite easily through the gel as compared to the larger ones.  When stained with a dye, the gel is then placed under UV light to observe the migration of DNA bands, which represent same-sized DNA fragment. By comparing the bands to the DNA marker ladder, we can determine the approximate size of the bands, and determine the sites where the restriction enzyme cleaved the plasmid.Mapping of plasmid is an integral part of molecular biology as they are used to develop and plan a cloning strategy as well as in DNA cloning. When a plasmid is cut by a restriction enzyme and run on a n agarose gel, bands of different cleaved fragments are obtained which are then compared to the standard size markers. Using this information, the sites on the plasmid at which each restriction enzyme cuts can be located.The aim of this experiment is to determine whether plasmid DNA is cut into different sized fragments by using a variety of restriction enzymes- namely EcoR1, Pst1 and BamH1. The size of the fragments was estimated by plotting a semilogarithmic graph of the distance travelled by each fragment (mm) to the fragment size (in kilobase pairs), which was used to map the plasmid. Materials and MethodsStep 1: Cleaving and digestion of unknown plasmid DNA with restriction enzymesThe unknown plasmid DNA is cleaved using a variety of restriction enzymes; namely EcoR1, BamH1, and Pst1; as follows:Tube (i)- Eco R1Tube (ii)- Pst 1Tube (iii)- Bam H1Tube (iv)- Bam H1 & Pst 1Tube (v)- Bam H1, Pst 1 & Eco R1Single digestions of the unknown plasmid DNA were carried out using 5uls of plasmid DNA, to which 3uls of 10X enzyme buffer were added. To this single digest, 3uls of appropriate enzymes were added and the total volume was made to 30uls by adding 21 uls of sterile water.Similarly, double and triple digestions were carried out by adding 3uls of 10X enzyme buffer, 3uls of appropriate enzymes to 5uls of plasmid solution. To makes the final volume amount to 30uls, 20uls of sterile water was added to the double digest tube while 19uls of sterile water was added to the triple digest tube.Step 2: Agarose Gel Electrophoretic analysis of unknown plasmidThese digestions were run on 1% agarose gel; prepared by weighing out 1.5g of agarose and mixing it in a solution of 15ml of 10X TBE buffer and 135ml of water in a 250ml flask. The mixture was swirled and heated in a microwave oven for 3mins until the agarose dissolved.  A solution of 600mls of 1X TBE buffer, prepared by mixing 60ml of 10X TBE buffer with 540ml of distilled water, was poured carefully into the electrophoresis tank. The gel was then poured carefully into the mould; removing any air bubble that may have formed with the help of a pipette tip. The mould was set aside, and care taken to not disturb it until solidified into a milky white and translucent gel. Once solidified, the well former was removed carefully to not break the wells.Step 3: Loading the samples into the agarose gel Each digestion sample, stained with fluorescent dye, ethidium bromide, was loaded into the slots of the well with the molecular size marker loaded into the first slot, followed by the prepared digestion samples. The gel was electrophoresed at 100V for 3 hours.Step 4: Visualization of DNA bandsThe DNA bands were visualized using a trans-illuminator with Ultraviolet light source. ResultsTo create a possible plasmid map, single, double and triple digestions were performed using EcoR1, Pst 1 and Bam H1 restriction enzymes. The result obtained are shown in the Figure 1.1. Lamda Hind III (kb)9.46.74.42.32.00.6                 (i)       (ii)      (iii)      (iv)      (v)      (vi)Figure 1.1: Electrophoretic analysis of digested plasmid X cleaved with a three types of restriction enzymes.(i)Lambda Hind III marker; (ii) DNA fragment cleaved with EcoR1; (iii) DNA fragment cleaved with Pst1; (iv) DNA fragment cleaved with BamH1; (v) DNA fragment cleaved with BamH1 and Pst1; (vi) DNA fragment cleaved with BamH1, Pst1 and EcoR1.To determine the size of the plasmid DNA fragments cleaved by the restriction enzymes, a logarithmic graph (log10) was plotted using a set of known size marker, Lambda Hind III.Graph 1.1: The above semilogarithmic graph displays the distance travelled (in millimetres) through the electrophoretic gel to the fragment size (log kilobase pairs) of the cleaved plasmid DNA fragments, cut by Hind III digested lambda size markers.From the graph, R-squared value and equation of the graph were calculated as follows_y=30.422e-0.039xR² = 0.9667Using this equation, the size of the DNA fragments cleaved by the three restriction enzymes- BamH1, EcoR1 and Pst1- were calculated using this equation. The results obtained are summarised in Table1.1.?Hind3 (Marker)Distance moved in gel (mm)?Hind3 (Marker) (kb)Plasmid XEcoR1 (kb)Plasmid XPst1(kb)Plasmid XEcoR1Plasmid XBamH1(kb)Plasmid XBamH1Pst1(kb)Plasmid XBamH1Pst1 EcoR1(kb)369.45.475.585.583.563.56416.7   3.171.6947.54.4    1.10561.52.3     652.0     1050.6   = 6.73= 6.355Table 1.1: The above table displays the results obtained from the single, double and triple enzyme restriction digest of plasmid.The distance travelled by each DNA fragment is measured and used in calculating the number of base pairs of each fragment.DiscussionA 1%agarose gel was used to run the digestions and after staining it with ethidium bromide solution, the cleaved DNA fragments were visible under a trans-illuminator using an Ultraviolet light source.The unknown plasmid DNA was cut into small fragments of varying sizes at different sites of the plasmid by each of the three restriction enzymes; causing them to travel varying distances at different rates during gel electrophoresis, as seen in Figure 1.1. The restriction enzyme marker Hind III cleaved the plasmid DNA into several fragments (6 fragments), causing the fragments to travel a total distance of 105 mm through the agarose gel. Similarly,the single digestions of Eco R1, Pst1 and BamH1 cut the plasmid DNA into one fragment, yielding single bands, each of approximately 5.47 (EcoR1) and 5.58kb, travelling through the gel to a distance of 44mm and 43.5mm respectively.  In the double digestion involving the restriction enzymes BamH1 and Pst1, the plasmid DNA was cleaved into 2 fragments, yielding 2 bands of sizes 3.63kb and 3.17kb at 54.5mm and 58mm respectively. Similarly, in triple digestions, 3 fragments were obtained with band sizes of 3.56 kb, 1.69 kb and 1.105kb, at 55mm, 74mm and 85mm respectively.From the Table 1.1, the size of Plasmid X is approximately calculated to be 6.355kb; or having approx. 6355 base pairs. This has been calculated using the values obtained in triple digest as the fragments are smaller in size and provide more accuracy. It is a known fact, that DNA fragments cleaved by variety of restriction enzyme move at different rates a varying distance through the gel. Smaller fragments move much quicker than larger fragments through the gel easily, for a longer distance. Thus, the smaller fragments will provide more accurate results.To calculate the size of plasmid X, the distance travelled (in mm) by each fragment in each digestion was calculated and was put in the equation (as seen in equation 1). This gave the size of the DNA fragments in kilobase pairs. The total size of the fragments in double and triple digestions should equal the fragment size in single digest. However, as this was not obtained due to small inaccuracies, the more accurate size was taken from the triple digest, as stated above.The single digestions of Eco R1, Pst1 and BamH1 cut the plasmid DNA into one fragment, yielding single bands, whereas, in the double digestion involving the restriction enzymes BamH1 and Pst1, the plasmid DNA was cleaved into 2 fragments, yielding 2 bands. Similarly, in triple digestions, 3 fragments were obtained yielding 3 bands when viewed under UV light.With the given data, there are two equally plausible maps which fit the obtained results, as shown in Figure 1.3.       0Bam H1 cuts at 0 base pair positionEcoR1 cuts at 4665 base pair positionORcuts at 5250 base pair positionPst1 cuts at approx. 3560 base pair position   6.3kb6.3kb       Figure 1.3: Two possible plasmid X maps of a 6.3kb Plasmid Plasmid X has a single site for each of the restriction enzyme, as each restriction enzyme seems to have cut the plasmid at one site, resulting in the formation of a single band. In double digest, two bands are observed due to cutting of plasmid DNA by BamH1 and Pst1. These are first marked on the plasmid map with a distance of 3630 bp and 3170 bp separating them; however, it remains unknown as to which enzymes cuts at either distance. With the addition of EcoR1 enzyme, the second band breaks further into two smaller bands each of 1.69 and 1.105kb, confirming that digestion by EcoR1 has occurred. However, since the exact location of the enzymes is undetermined, it is possible that the site cut by EcoR1 may either lie close to the site cut by BamH1 or near the site cut by Pst1; resulting in two possible plasmid maps as seen in Figure 1.3.To distinguish between the two possibilities and locate the EcoR1 cleaving site on the plasmid, it is possible to carry out further restriction enzyme digestions using double digest. There are two possible digestions. The first digestion could involve restriction enzymes BamH1 and EcoR1, while the second digestion could involve Pst 1 and EcoR1.For example, as seen in Figure 1.4, if double digestion using BamH1 and EcoR1 were performed, the following possibilities could be obtained. If the size of the bands obtained through gel electrophoresis coincide with the value shown in figure, it is possible to locate the exact position of EcoR1, such as if the size of EcoR1 band was calculated as 1.105kb, the location of EcoR1 could be seen as in Figure 1.4 (a); close to BamH1. Similarly, if the size was calculated to be 1.69kb, the location of EcoR1 would be close to Pst1.  1690 bp (1.69kb) kb1.1056.3kb4665 bp (4.665 kb)1.691.105Figure 1.4: Double Digestion of plasmid DNA using BamH1 and EcoR1 restriction enzyme. The accuracy of the map is dependent on how precisely the exact size of a fragment is determined.Restriction enzyme usually have a short target sequence of approximately 4-8 base pairs; which vary in frequency. The restriction enzymes cut DNA at specific target sites or sequence. This means that a restriction enzyme will always cut the DNA molecule into same set of fragments (https://www.ncbi.nlm.nih.gov/books/NBK21116/). If the DNA is cut by the restriction enzyme several times, the more distance it would travel through the gel during electrophoresis. Smaller fragments move much quicker than larger fragments through the gel easily. In order words, larger fragments will travel a shorter distance as compared to the smaller ones and will usually be seen at the top of the gel. Thus, the smaller fragments will be more accurate(book).Moreover, the size of large fragments in a gel cannot be differentiated. For example, it would be difficult to differentiate between a 10kb fragment with 9kb fragment, as they both look the same in the gel. Hence, larger fragments will always be less accurate than the smaller fragments.To increase the accuracy of the Plasmid X map, the agarose gel and buffer concentration can be increased. The number of enzyme digestions can also be increased to further improve the accuracy of mapping. A technique of optical mapping has taken over the physical mapping of plasmids due to its inaccuracies. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC19846/)Agarose Gel Electrophoretic Comparison of the two plasmid DNA digestionsLamda Hind III (kb)9.46.74.42.32.00.6      (a)                  (i)      (ii)      (iii)      (iv)       (v)       (vi)(b)                  (i)    (ii)    (iii)     (iv)       (v)       (vi) Figure 1.2: A side-by-side comparison of two gels as viewed under ultraviolet light.Figure 1.2(a) shows a professor- run gel electrophoretic analysis of digested plasmid X cleaved with a three types of restriction enzymes, while Figure 1.2(b) shows a student-run gel electrophoresis. (i)Lambda Hind III marker; (ii) DNA fragment cleaved with EcoR1; (iii) DNA fragment cleaved with Pst1; (iv) DNA fragment cleaved with BamH1; (v) DNA fragment cleaved with BamH1 and Pst1; (vi) DNA fragment cleaved with BamH1, Pst1 and EcoR1.Figure 1.2(a) shows the correct result for single, double and triple digestion of the unknown plasmid DNA cleaved by the restriction enzymes BamH1, Pst 1 and EcoR1, while mistakes have been incorporated in the student-run gel.In the student-run gel (Figure 1.2b), some mistakes have been made in terms of wrong pipetting, dilution and measurement errors resulting in human experimental error. Mistakes in measurement may have occurred due to wrong pipetting while measuring the amount of plasmid DNA and restriction enzymes put into Eppendorf tubes. Precise quantities of enzymes are required for a complete digestion to take place; otherwise impartial digestion of unexpected patterns may develop resulting in error. Moreover, inaccuracies may have been incorporated during the calculation process while manually measuring the distance moved by each fragment through the gel. Because of this error, the values varied marginally from the original base pair number. Error may also result due to contamination of pipetting tubes, Eppendorf tubes and other materials used in the experiment.Another error can be seen on the gel when viewed under ultraviolet light. The bands cannot be seen as a result of not enough staining or due to a very diluted sample of plasmid DNA. Faintness of bands may also be due to a less strong UV light source. The student- run gel serves as a good example of how accuracy in pipetting and measurements are important in molecular biology, especially when dealing with amounts in microlitres (ul).ReferencesMcb.berkeley.edu>courses>slides— uc Berkeley mcb laboratory report guidelinesAL, C., 1936. B IOLO GY. Nature, 137, p.444.www. =scribd.com/doc/314354682/dna-restriction-analysis-lab-reporthttps://link.springer.com/article/10.1186%2Fs13059-018-1398-0https://www.ohio.edu/plantbio/staff/showalte/MCB%20730/MCB%207300%20Lab%204%20-%20Restriction%20analysis.pdfhttp://users.stlcc.edu/departments/fvbio/Bio219_Lab_Manual/Lab%2012%20PLASMID%20MAPPING.pdfhttp://www2.southeastern.edu/Academics/Faculty/jtemple/486/experiment%202.pdfhttps://www.ncbi.nlm.nih.gov/books/NBK21498/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC19846/