The molecular biology industry is vastly increasing due to the increasing use of restriction enzymes in the generation of fragments which can further be used in gene analysis such as RFLP and gene cloning techniques. In this practical, restriction enzymes EcoRI, BamHI, and HindIII were used to cut up λ DNA as both single enzymes and also a combination of the different enzymes, the mixture of the DNA and the enzymes were then loaded unto 0.8% agarose gel in order to separate the fragments whose sizes could be measured in order to draw a restriction map. This practical however was not successful and therefore appropriate fragment sizes could not be worked out.
Since its first isolation (Smith et al 1970) restriction enzymes (RE) has become a major tool utilised by researcher to cut genomic DNA into smaller fragment useful for investigating gene alignment, activity and expression through restriction fragment lenght polymorphism [RFLP) (Hartwell et al 2003). RE originates from bacteria and serves as a form of immune defence against viral infection by digesting the viral DNA. These enzymes recognizes a specific sequence of bases anywhere within the genome and then serves two covalent bond in the sugar-phospahte backbone at a particular position within or near that sequence. Over 3000 restriction enzymes have been identified and classified, ranging from type I-III based on subunit constitution, cleavage position, sequence specificity and cofactor requirements. Type I RE are quite complex as they cuts DNA at random locations as far as 1000bp from the recognition site. They composed three different subunits M (modification), R (restriction) and S (specificity) that form multifunctional enzymes with restriction, methylase and ATPase activities. Type III RE are hetero-oligomeric that composed of two subunits (res and Mod) , With similarity to type I enzyme they are also ATP dependent (encoded by Res) , the Mod possess sequence specific methylation restriction and DNA dependent nucleoside triphosphatase activities . These sequence recognises the short 5-6 bp long asymmetric DNA and subsequently cleave DNA at non-specific loci adjacent to the site 25-28 bp nucleotides 3′ ( ). Type II RE are predominantly used in biotechnology for DNA analysis and gene cloning, since 1997 over 1000 new RE has been identified. They recognise short sequence 4-8bp and cleave within or close to proximity of the recognition sequence in the presence of magnesium. In this investigation Three type II RE ( HIND III, BAMH I, and ECORI) are used in a series of single, multiple and triple enzymatic digest using lambda (λ) DNA as a substrate. Bacteriophage λ DNA is a linear molecule containing 4800kb that originates from phage Lambda virus. It's a well known substrate for RE digestion. All three enzymes were isolated from bacteria . EcoRI from Escherichia .coli is specific for cutting GAATTC and produces sticky ends with the 5' end of DNA ( ), while HindIII isolated from Haemophillus influenza cleaves between the Adenine- Adenine (AA) which causes 5' ends to overhang ( ) BamHI isolated from Bacillus .Amiloliquefaciens is an enzyme that's highly useful; because of its ability to leaves the GATC hanging after enzymic cleavage thus able to attach to other enzymes (Becker et al 1990). These enzymes are used to digest λ DNA and evaluated gel electrophoresis
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Fast digest enzymes EcoRI®, BamHI® and HindIII®, 10x TE buffer obtained from fermentas. Deoxyribonucleic acid methylated, bacteriophage (Lambda Phage) from E. coli host strain W3110 (10mM Tris-Hcl at a pH of 8.0 and 1mM EDTA) and HindIII DNA ladder obtained from SIGMA-ALDRICH. Distilled H20 (dH2O) ,0.8% agrose gel supplied by Kingston university. 7 epoendorf tubes were labelled according to the restriction enzyme digest reaction. First three (single digest assay) tube 1-3 contained 11µl of dH2O , 2µl of λ DNA, 2µl of 10x TE buffer and cleavage were initiated by addition of 5µl of fast digest EcoRI, BamHI and HindIII respectively to a final volume of 20µl in each tube. Tube 4-6 (double digest assay) contained 6µl of dH2O, 2µl of λDNA, 2µl of 10x TE buffer and double digestion was initiated by addition of 5µl of EcoRI/BAMHI each, EcoRI/HindIII, and BamHI/HindIII respectively to a final volume of 20µl in each tube. The final tube 7 ( triple digest assay) contained 1µl of dH2O, 2µl of 10X TE buffer, 2µl of λ DNA and triple digestion was initiated by addition of 5µl of EcoRI, BamHI and HINDIII each. The tubes were centrifuged and placed in a 650C water bath for 5 minutes (error as opposed to the 37 0C). 2µl of loading dye was added to the tubes and incubated again at 650C for 10minutes. The tubes were recentrifuged alongside a tube of λ DNA HindIII digest as a control to eliminate the bubbles. The 22µl of tube content were loaded into the wells of the pre-prepared 0.8% agarose gel and run at 90volts for 40 minutes . gels were viewed under ultraviolet transilluminator and image obtaineWere possible the DNAfragment band were evaluated by scintillation counting.
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Figure 1: Schematic diagram of agrose gel separation of λ DNA digested by three different restriction enzyme EcoRI, BamHI, and HINDIII. First three lane show single digest of DNA respectively . lane 4-6 contains double digest reaction EcoRI/BamHI, EcoRI/HindIII and BamhHI/HindIII repectively. Lane 7 contains triple digest reaction with all three enzymes EcoRI/BamHI/HindIII. Lane 8 contain HindIII DNA marker.The fragments were separated by agarose gel (0.8%) electrophoresis and the visualised under ultraviolet transilluminator. L-(LANE)gel16.jpg From the agrose gel picture obtained (fig 1) it can be seen that lane 1(EcoRI)presents very faint 5 bands however it and it seems like the band has not separated down the well properly in order to distinguish individual band, the same can also be said for lane 2 (BamHI) as only 3 thick distinct band can seen opposed to 5 that was expected. Lane 3 (HindIII) present 7 fragment band although also faint the 6 bands can be seen instead of 7 expected. Lane 4 -6 containing the double digests as mention in the methodology, did not show bands that were distinctive as some of this bands were suppose to correspond to two or three more band from the single digest however some of the bands in these lanes appears thicker and brighter probably indicating double digest of both enzyme cutting at the same site.. Lane 7 showing band for the triple digest were very unclear bands were too close to each other and unable distinguish they did not separate properly. It dye runs can be seen on al the first band aside from the 8th lane which was the Ladder and all band in this were separated properly. Figure 2 is showing a restriction map obtained from a website which shows all the points all three enzymes were suppose to cut and how many band was suppose to show on our gel in order to draw a restriction map.
Figure 2 : schematic Map of λ DNA digested with three different enzyme BamHI (GGATCC recognition site found in λ DNA at 5 locations), EcoRI (GAATTC-5 location) and HindIII ( AAGCTT-7 location ) also showing size of each fragment size. Map obtained from http ://ww w.apsnet. org/edcenter/K-12/TeachersGuide /Plan t Biote chnol ogy/Pa ges/Act ivity3.aspx and modified.
This experiment was generated to initiate series of single, double and triple restriction enzymatic digest on bacteriophage DNA lambda, in order to determine how all three enzymes will cleave and digest DNA λ. The end result from this experiment was unsatisfactory; however this was partially expected due to the number of errors that occurred during methodology process. Ideally if the experiment had run smoothly and clear bands were obtained the band on each lane of the gel picture in Fig 1 would be measured (distance travelled from well) and each band (fragment) size would have been extrapolated from a standard curve drawn using the DNA HindIII ladder, and a proper genome map would be constructed. However the results obtained were inadequate hence could not be done. It is clear from first bands on each lane of the gel picture that lack of pippetting skill into gel wells has notably affected the visualisation of the bands. This was probably done by digging pipette tip into the well thus allowing significant amount of the sample to run under the gel. Optimality and maintaining the right environment for each reaction is crucial for this procedure, however this was breach when the samples were accidentally placed at 65oC to initiate enzyme digestion. Consequently causing denaturation of enzymes which might be the reason why the fragment were not separated properly. Although fermentas product sheet state enzyme inactivation at 80oC. Another process that might have affected the result negatively is delayed incubation time (10min) before gel electrophoresis, which could have affected breakage hydrogen bond holding the ends of the linear DNA together in a circle.