Extrachromosomal DNA Molecules - Essay Example

? Plasmid mapping Introduction: Plasmids are the extrachromosomal DNA molecules, and are mostly double –stranded, circular and covalently closed molecules, varying in size from 1 kb to 200 kb. (Sambrook and Russell 2001). They are found in many bacterial species. They replicate independently and use a variety of mechanisms to maintain their copy number. They contain the gene codes for the enzymes that are important for the bacterial hosts. The plasmids act as vectors in the molecular biology experiments. The vectors are the carrier DNA molecules into which the foreign gene of interest is inserted and expressed in the host. This DNA is now called as recombinant DNA. These vectors replicate inside the host cell along with the inserted DNA. These vectors are of two types: expression vectors (expression of the cloned gene to give the desired protein) and cloning vectors (produce millions of copies of cloned DNA). (Sambrook and Russell 2001). Restriction endonucleases are the enzymes that cut the DNA at the specific sequences. There are about 200 different restriction enzymes. (Brown 1995). The most common restriction endonucleases are EcoR1, BamH 1 and Pst1. All these restriction enzymes have sticky ends. The recognition sites for these restriction enzymes are as follows: EcoRI recognition site = G|AATTC Bam H1 recognition site = G|GATCC C TTAA |G C C T A GIG and Pst 1 recognition site = CTGCAIG GIACGTC (Siwach and Singh 2007). The pieces of DNA that remain after the digestion with the restriction enzymes are called as restriction fragments. Each restriction enzyme has a unique code and it cuts the DNA into fragments with either sticky or blunt ends. A restriction map gives us the location where the restriction enzyme cuts the DNA. This restriction digestion is used for two purposes: Restriction mapping and specific DNA cleavage for the production of new constructs. The restriction mapping is used to identify the plasmids. The number of DNA fragments and the size of the DNA fragments depend upon the action of the restriction enzyme. These DNA fragments thus obtained are separated using the Agarose gel electrophoresis. Gel electrophoresis is the most powerful technique for separating the biomolecules. The DNA are negatively charged particles that are attracted towards the opposite charge under the influence of electric field. Here the agarose gel is the solid matrix. The solid matrix controls the rate of migration of the molecules based on the size of the particles and the concentration of the gel. The buffer is a mixture of organic and inorganic salts that helps to conduct the electric current between the positive and negative terminals. To visualize the DNA bands present in the gel, stains such as methylene blue and ethidium bromide are used. In our experiment we use ethidium bromide that fluorescence’s under the UV light. Ethidium bromide intercalates between the base pairs of DNA and fluorescence when exposed to the light of 250 – 300 nm. (Sambrook and Russell 2001). Materials and method: The materials are the same as mentioned in the practical handbook. Restriction mapping consists of three important steps. They are restriction enzyme digestion, agarose gel preparation and sample loading. 1) Restriction Enzyme digestion: The unknown plasmid sample is taken and they are digested using the restriction enzyme. The standard concentration of the plasmid DNA is 1 ?g/ 5 ?l. In order to standardize the plasmid and to enhance the enzyme reaction, 2 ?l of enzyme buffer is added to the digest. 1 ?l of the enzyme is added to the sample. The volume of the digest is made upto 20 ?l using the sterile water. The composition of the digest is as follows: For this Restriction digestion, BameH1,Pst1 and EcoR1 restriction enzymes were used. The final volume of each restriction digest was 20 microlitres. ? Hind3 (Marker)-Distance moved in the gel mm Plasmid DNA Enzyme 10Xenzyme buffer Sterile water Total EcoR1 5µl 1µl 2µl 12µl 20µl Pst1 5µl 1µl 2µl 12µl 20µl BamH1 5µl 1µl 2µl 12µl 20µl BamH1&Pst1 5µl 1µlBamH1 1µlPst1 2µl 11µl 20µl BamH1&Pst1&EcoR1 5µl 1µlBamH1, 1µlPst1 1µlEcoR1 2µl 10µl 20µl After the final step, the contents of the tube are gently mixed by flicking the samples with the fingertip. If drops are found sticking to the sides then they are brought together by using the micro centrifuge. The digest is incubated at 37 oC for one hour. (most of the restriction enzymes works best at 37 oC). To make the sample dense 4 µl of the blue loading dye is added to all the samples. The samples are mixed gently by flicking. 4 µl of the blue loading dye is added to the size marker also. Now the sample is ready for loading in the gel. 2) Agarose gel preparation: The gel mould is taken and the two sides of the mould are tapped with the masking tape and the comb is inserted at the appropriate height and distance from one end of the gel. Care should be taken not to place the comb too depth inside the casting. 1.5 gram of agarose is weighed and added to the 250 ml of conical flask. To this 15ml 10XTAE buffer and 135ml of distilled water is added. The agarose is heated in the oven until the agarose dissolves in the buffer. Care should be taken not to over boil the agarose. The agarose solution is taken out of the oven and kept away. Add 15 ?ls of the fluorescent dye ethidium bromide to the agarose solution and swirl to mix the dye gently. Pour the agarose on the mould. If there are any bubbles present, remove them using the micro pipette. After the gel has set and appears as milky white translucent gel, remove the combs and tapes on both ends carefully. Place the gel onto the electrophoresis set up with 700 ml of 1XTAE buffer. (This is prepared by adding 60 ml of 10XTAE to 540ml of distilled water). 3) Sample loading: The wells in the gel are marked as 1, 2,3,4,5 and 6. The first well is loaded with 20 µl of the size marker (the size marker is lambda DNA digested with HindIII) and the rest of the wells are loaded with the digested samples. The gel was electrphoresed at 100 v for 3 hours. The gel is then taken from the tank and the DNA bands are viewed under the UV transilluminator. The gel is photographed and the distance migrated by the DNA bands in each well are found. Results and Discussion: Figure 1: Photograph of the Gel. Lane 1 = Size marker, Lane 2 = Plasmid X digested with BamH1, Lane 3 = Plasmid X digested with Pst1, Lane 4 = Plasmid X digested with EcoR1, Lane 5 = Plasmid X digested with BamH1 and Pst1, Lane 6 = Plasmid X digested with EcoR1, BamH1 and Pst1. Table1: The size of the plasmid DNA fragments obtained in the digestion. ? Hind3 (Marker) -Distance moved in the gel mm ? Hind3 (Marker) kb Plasmid X BamH1 (kb) Plasmid X Pst1 (kb) Plasmid X EcoR1 (kb) Plasmid X BamH1 Pst1 (kb) Plasmid X BamH1 Pst1 EcoR1 (kb) 14 9.4 6.8 6.8 6.8 3.8 3.8 18 6.7 3.0 1.6 21 4.4 1.4 29 2.3 31 2.0 45 0.6 Figure 2: log of lambda Hind111 marker size against migration distance From the results it is found that the plasmid is of molecular weight 6.8 kilo base pairs. When the plasmid X was digested with only one restriction enzyme, it produced a single band. This concludes that there are only one restriction site for EcoR1, BamH1 and Pst1. When the plasmid X was digested with BamH1 and Pst1, it produced two bands of 3.8 kb and 3.0 kb indicating that the two restriction sites are present far away from each other. Similarly when the three restriction enzymes EcoR1, BamH1 and Pst1 were used for the digestion, three bands were obtained with the bands at 3. 8, 1.6 and 1.4 kb respectively. This confirms that the three restriction sites are present at a distance from each other. From the above results the restriction mapping for the plasmid X can be drawn Answers to the Questions: 1. The total base pairs of the plasmid X is 6.8 kb. This can be concluded from the bands obtained after each digestion. In the first digestion, at the lane 2, the total kb is 6.8 for a single band. The same results were obtained for the three restriction enzymes when used separately. In the fifth lane, the two DNA bands were seen with the base pairs of 3.8 kb and 3.0 kb respectively. From these results it is concluded that plasmid X is of 6.8kb. (Passarage 2007) 2. In the gel, only one DNA band was seen for the restriction digestion of the enzyme EcoR1 in the lane 2, BamH1 in the lane 3 and Pst1 in the lane 4. Similarly when these three enzymes were used together, three bands were seen on the lane 6. Thus it was found that there are only one restriction site for the enzymes EcoR1, BamH1 and Pst1 in the plasmid X. 3. The restriction mapping of the plasmid X is as follows: If the total base pair is 6.8 kb, we start with the assumption that the first cleavage site is present at the first base pair. Let us assume that Bam H1 has the cleavage site at the first base pair. According to the results obtained from the lane 5, two bands of 3.8 kb and 3.0 kb are present in the plasmid. Then the restriction site of the Pst 1 should be 3.8 kb away from the first restriction site BamH1. When three restriction enzymes were used together, three bands were produced with 3.8kb, 1.6 kb and 1.4 kb. From this data we can confirm that the EcoR1 restriction site is present only next to the Pst 1 restriction site and the distance between the BamH1 and Pst 1 is constant. (Siwach and Singh 2007); (Kruezer and Massey 2008). From these conclusions the restriction mapping for the plasmid X was drawn. References: Brown, TA 1995, Gene Cloning : An introduction, Chapman and Hall. Kruezer , H and Massey, A 2008, Molecular Biology and Biotechnology: a guide for teachers, ASM Press. Passarage, E 2007, Color Atlas of Genetics, Thieme. Sambrook, J and Russell, DW 2001, Molecular Cloning: A Laboratory manual, CSHL Press. Siwach, P and Singh, N 2007, Molecular Biology: Principles and Practice, Firewall Media. Read More