This paper describes how Recombinant DNA can be created in a laboratory using simple Lambda DNA and the pUC18 plasmid. This study involved digesting Lambda DNA with the restriction enzyme EcoRI, and then ligating the resulting DNA fragments into the pUC18 plasmid. The recombinant molecules were characterized by electrophoresis, and then transformed after introduction into competent DH5 alpha cells. The cells were then placed on agar plates that contained both ampicillin and X gal, which allowed an easy identification of the plasmids that contained the Lambda DNA inserts, via their white color. The colonies were further digested with the restriction enzymes EcoRI and a combination of EcorRI and BamHI to determine the size of the DNA fragments with gel electrophoresis. The final step was to perform Southern blot procedures to definitively identify the specific fragments that contained the Lambda DNA.
All DNA is composed of four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C). These nitrogen bases are found in pairs, A pairing with T and G pairing with C. The specific sequence of the nitrogen bases can be arranged in an infinite number of ways. This infinite number of possible sequences is what creates diversity in organisms. (Casey. 1992) DNA is the foundation for various proteins, and it is these proteins that cause certain characteristics to be expressed in organisms. Changing the DNA sequence, changes the way in which the protein is formed. This leads either to the expression of a different protein, or an inactivation of the present protein. (Watson. 1992)
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Recombinant DNA is a form of artificial DNA that is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences that would not normally occur together. This recombination controls which proteins are active, and thus, which genes are expressed or inactivated. (Bharathan. 2009) In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organism genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered.
Recombinant DNA technology is important in a wide variety of ways. Recombinant DNA technology is used in medicine to identify human genes whose mutations play important roles in relation to diseases, and in treating those diseases through gene therapy. (Campbell. 2005) Recombinant DNA technology also can be used in agriculture to modify plants in beneficial ways. The starch and protein levels of seeds can be modified to increase successful seed germination. Plants can also be modified to have increased pest of virus resistance. Nutritional value of plants can also be modified by increasing the Vitamin content of the plant. (Alberts. 2009)
Recombination of DNA is achieved through a series of steps. First, the DNA of interest must be isolated from an organism. The DNA is then cut with restriction enzymes. Restriction enzymes are a special class of nucleases that cut DNA only at particular places in the DNA sequence. (Alberts. 2009) These cut fragments of DNA must be spliced into a plasmid. A plasmid is a small circular DNA molecule that contains replication origins and can replicate independently. (Alberts. 2009) Once the DNA fragment is successfully spliced into the plasmid, the new piece is referred to as the recombinant DNA. This recombinant DNA is then placed back into a host's cell for transformation to take place. Transformation is when the new gene is incorporated into the DNA of the host's own chromosomes, making clones of the recombinant DNA. (Griffiths. 1996) The newly cloned DNA can then be recovered from the host, and analyzed by several methods to definitively identify the recombinant DNA. One method is to use a plasmid that contains a gene that is resistant to antibiotics or contains the lacZ reporter gene, which produces Î²-galactocidase and reacts with X-gal to create a blue color. If the plasmid contains the lacZ reporter gene, and it is not interrupted by an insertion of foreign DNA, then it will continue to produce Î²-galactocidase, and this will interact with the X-gal in the agar plates, and result in a blue color. White colonies are the result of an interruption of the lacZ gene and thus indicate complete ligation of foreign DNA. Another method for identification of recombinant DNA is to use gel electrophoresis to try to determine the specific fragments that are recombinant. To definitively verify the gel electrophoresis analysis, you have to perform a Southern blot on the samples. Southern blot is a method to transfer the fragments from a gel to some sort of solid support sheet so that hybridization can take place. The fragments of DNA are subjected to special buffers that break down the double helix formation, leaving only single stranded DNA. This is imperative to allow a probe to be introduced that binds to the single stranded DNA and labels the strand so that specific sequences can be easily detected. (Bharathan. 2009) The encoded gene product can then be used for research of other commercial purposes.
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The purpose of this study was two-fold. The first objective was to learn the techniques of how to create a recombinant DNA molecule using Lambda DNA and the plasmid pUC18. The second objective was to digest the recombinant DNA and analyze the resulting fragments to identify which fragments contain recombinant DNA.
Materials and Methods:
Lambda DNA and pUC18 were provided by Dr. Bharathan. Micro centrifuge tubes were obtained and labeled. Lambda DNA (20 ul) was placed into tube #1. pUC18 (20 ul) was placed into tube # 2. Restriction enzyme buffer (EcoRI) was obtained from Dr. Bharathan, and 40 ul of EcoRI was added to each of the tubes, 1 and 2. The tubes were mixed gently and incubated @ 37â° C for 60 minutes. After the incubation period, 10 ul of the mixtures were taken from the tubes and placed into two new micro centrifuge tubes. To the new tubes, 10 ul of DNA sample buffer was added. Each new tube was mixed gently. Both of the new tubes containing restriction enzyme and sample buffer were labeled with an "E" to indicate that EcoRI had been added, and placed into a -20â°C freezer until next laboratory session.
Samples of Lambda DNA and pUC18 that had been cut with EcoRI and stored in the freezer were obtained. A new micro centrifuge tube were acquired and labeled with an "L". To this new micro centrifuge tube, 10 ul of pUC18 that had been cut with EcoRI and 40 ul of Lambda DNA that had been cut with EcoRI were added and gently mixed together. The tube was placed into a 70â°C water bath for exactly 10 minutes. Immediately after the water bath incubation, the tube was transferred to ice for 10 minutes. Next, 50 ul of DNA ligase buffer was added to the tube, and mixed gently. The tube was incubated at room temperature for 1 hour, and then placed into a -20â° freezer until next laboratory session.
The micro centrifuge tube labeled "L" was retrieved from the freezer and placed on ice, and allowed to sit for 5 minutes. Sample "L" (10 ul) was removed and placed into a new micro centrifuge tube labeled "L-E". To the "L-E" tube, 10 ul of DNA sample buffer was added, mixed, and placed back into the -20â°C freezer. To the tube labeled "L", 350 ul of competent DH5 alpha cells (obtained from Dr. Bharathan) were added, and gently mixed. This mixture was allowed to incubate on iced for 20 minutes. After the 20 minute incubation, the tube was transferred to a 37â°C water bath for 5 minutes. The tube was removed from the water bath, and 0.8 ul of plain nutrient broth was added to the tube, and the tube was placed back into the 37â°C water bath for 45 minutes. The tube was removed from the water bath and mixed gently, and 250 ul of the cells were spread onto an agar plate that contained Xgal and Ampicillin (provided by Dr. Bharathan). The agar plate was allowed to sit at room temperature until all the liquid had been absorbed. The plate was inverted and incubated @ 37â°C for 12-24 hours
Three screw-cap tubes were acquired and ~ 15 ml of nutrient broth was added to each tube. The tubes were marked "B" for blue colonies, and "W1" and "W2" for white colonies. Colonies were chosen from the agar plate. One blue colony was chosen and added to the screw-cap tube marked "B", while two separate white colonies were chosen and one white colony was incorporated into the tube marked "W1" while the other white colony was incorporated into the tube marked "W2". These tubes were then placed in a hot water bath, shaker overnight.
Six micro centrifuge tubes were obtained and labeled so that there were two tubes for each, "B", "W1", and "W2". Gently mix each of the screw-cap tubes, and place 1.5 ml of each of the cultures into the respective micro centrifuge tubes. All of the micro centrifuge tubes were centrifuged @ 2,000 RPM for 8 minutes. The supernatant was carefully discarded, and a Kim-Wipe was used to carefully dry out the lid and sides of the micro centrifuge tubes. The tubes were turned upside down and the pellet allowed to dry for 2 minutes. Lysis buffer (400 ul) was added to each tube and the pellet was resuspended by gently tapping each tube with a finger. The tubes were incubated @ room temperature for 10 minutes. SDS-NaOH (400 ul) was added to each tube and gently mixed. The tubes were incubated on ice for 15 minutes. After the incubation, Ammonium Acetate (300 ul) was added to each tube and gently mixed. The tubes were incubated on ice for an additional 15 minutes. The tubes were then centrifuged @ highest speed for 10 minutes, and the supernatant was poured from each tube was poured into new respective micro centrifuge tubes. Isopropanol (1 ml) was added to each of the new tubes, and gently shaken. All tubes were placed into -20â°C freezer for 30 minutes and then centrifuged @ highest speed for 10 minutes. The supernatant was poured off, and 1.1 ml of Isopropanol was added to the tube. The tube was incubated @ room temperature for 15 minutes, and then centrifuged @ 5000 RPM's for 10 minutes. The supernatant was carefully poured off, and the pellet was allowed to air dry. Sterile water (20 ul) was added to each of the tubes.
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New micro centrifuge tubes (8) were obtained and labeled. To tubes 1-4, EcoRI buffer solution (10 ul) was added, and to tubes 5-8, EcoRI + BamHI (10 ul) was added. Lambda DNA (10 ul) was added to tube 1 and 5. Blue plasmid (10 ul) was added to tube 2 and 6. W1 plasmid(10 ul) was added to tube 3 and 7, and W2 plasmid (10 ul) was added to tube 4 and 8. All eight tubes were placed into a -20â°C freezer. Gel electrophoresis analysis was conducted on the 8 samples using a 1% agarose gel, and a picture was taken.
The results as seen in Figure 1 show the various bands present after EcoRI digestion of Lambda, pUC18, and a combination of the two. (LE) Figure 2 shows the results from gel electrophoresis of nuclease digested plasmids from purified plasmids obtained after transformation. Figure 3 shows Southern blot data.
Recombinant Gel Pic.jpg
Figure 1: Gel electrophoresis of Lambda cut with EcoRI (Lambda-E), Puc18 cut with EcoRI (pUC18-E), and ligation of both. (LE)
Figure 2: Agar plate containing X-gal and Ampicillin showing growth of blue and white colonies.
Recombinant Gel 2.jpg
Figure 3: Gel electrophoresis showing products after restriction nuclease digestion.
EcoRI + BamHI
EcoRI + BamHI
EcoRI + BamHI
EcoRI + BamHI
Figure 4: Southern blot data, indicating the location of plasmids that have been labeled with Lambda probes.
Restriction nucleases have opened the doors to a whole new world of Recombinant DNA. It is now a fairly straight forward process to create Recombinant DNA in a laboratory. (Alberts. 2009) In this study, the restriction enzymes EcoRI and BamHI were used to cut Lambda DNA and pUC18. We see in Figure 1, that transformation has occurred. The lane labeled LE is a combination of the other two labeled lanes, Lambda-E and pUC18-E. When the lanes are compared to each other, it is evident that some piece of Lambda has been incorporated into the pUC18 plasmid, because many of the band fragments seen in the Lambda-E lane are not evident in the LE lane, which is a combination of the Lambda-E and pUC18-E samples.
Recombination was further analyzed by using agar plates that contained Xgal and Ampicillin. As seen in Figure 2, there were both blue and white colonies that grew on the agar plate. This indicates that the white colonies have been successfully transformed, because the lacZ gene that produces Î² galactosidase has been interrupted due to a successful ligation of foreign DNA, thus there is no interaction between the Î² galactosidase and the Xgal in the agar plates, thus producing no blue color, only white color. (Bharathan. 2009)
In Figure 3, the gel picture shows products of nuclease digestion. The particular results of this gel do not seem to be perfect as it would be expected to see band fragments of some sort in all of lanes 3-4 and 7-8, because these lanes contain samples from white colonies that should have some band fragments present due to the successful ligation as indicated by the white color. It appears that lanes 2-4 and lane 6 were digested by nuclease to the point that the result is many very small fragments as indicated by the large smears at the bottom of each of those lanes. In lane 7, we do see various band fragments fairly clearly. Based on the sizes of fragments from known Lambda digested with EcoRI, it appears that in lane 7 there are fragments at ~3530, 2686, and the even smaller band fragments are most likely a result of the combination of both restriction enzymes, EcoRI and Bam HI.
In Figure 4, we see Southern blot data that has been provided by Dr. Bharathan. This figure shows us the specific fragments that have had Lambda DNA incorporated into the plasmid. We see this indicated by red lines designated as "plasmid" in lanes 22, 23, 44, E3, and E4. This data supports that DNA can be separated, labeled and identified definitively by using Southern blotting techniques.
In conclusion, it is clear from the data, that obtaining recombinant DNA was successful, however to definitively identify the specific recombinant DNA, it would be good to repeat the gel that we see in Figure 3 to see if better results were obtainable, and to conduct a Southern blot on actual samples.
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