Plasmid Restriction Mapping - Lab Report Example

Plasmid Restriction Mapping Answers to the Pre-laboratory Questions 1) An example of a plasmid map is shown below. What is a plasmid map and why is it useful? Plasmid maps are graphical illustration of plasmids which shows the positions of the main identifiable markers on DNA such as gene, name of the plasmid and restriction enzyme sites. They show essential features of the plasmid and they are used to plan cloning strategy and confirm if the DNA clone has been constructed successfully. It is also used to in mutagenesis research, in DNA profiling in medicine to match potential organ donor and in the study of plants and wild animals populations (Harisha, 2007). 2) Enzymatic restriction of a plasmid can be a single digest, a double digest or a triple digest. What does this mean and how many DNA fragments are expected if each of the restriction enzyme of a plasmid only cuts the plasmid once? Single, double and triple digest determines the number and the relative positions of the restrictions enzyme sites. They are used to determine the relative positions and the number of restriction endonucleases sites cuts. The single restriction enzyme digest indicates the number of cut sites present. The double and triple cut sites show the relative cut sites to each other. If a single restriction site digests a circular molecule, it will result to one fragment, but a linear molecule will result in two fragments. A double digest will produce two fragments in a circular molecule and three fragments in a linear molecule. Also, a triple digest results in three fragments in a circular molecule (Tropp, 2014). 3) Why is the use of restriction enzymes an important tool for plasmid mapping? Restriction enzymes are used in plasmid mapping to determine the order of the restriction sites in a DNA. The restriction maps are used to analyze a plasmid or a cloned DNA fragment. The DNA is cut at different restriction enzyme sites, generating a map which provides information about the DNA being examined. The restriction enzymes are also use to confirm the specific DNA fragment, basing on the already identified restriction enzyme sites (Turner, 2000). 4) Describe the process of how you might map a plasmid using restriction enzymes. Restriction enzymes or endonucleases are used to cut the DNA strand into fragments, before performing gel electrophoresis on it. The DNA fragments separates according to their molecular weight as they move through the gel. Then mapping can be done, which can be on a linear or a circular DNA. It begins with determining the size of a single digestion lane on the gel, which is equivalent to the total size of DNA (Turner, 2000). The number of restrictions sites and the sizes of fragments are also examined. For example two restriction sites produce two fragments on a circular piece of DNA. The next step is to find the double digestion lane as some single lanes has double enzyme digestion. The single enzyme digestion which disappears in the double digestion lane must have the second restriction enzyme site within it. The different combinations of bands are then mixed and marched until the correct order has been established. The final sites must be consistent the results obtained from all the enzyme digestion (Tropp, 2014). 5) Have you read the Risk Management document for this practical and are you ready to comply with the recommended risk control measures? I have read the Risk Management document for this experiment and I am ready to comply with the recommended risk control measures Part 2: Discussion and answers to Questions: 1) A plasmid was cut using two restriction enzymes (EcoRI and HindIII), both individually and together. The samples were then run on an agarose gel, and the size of each fragment was recorded (see below). Use this information to answer the following2: Sample Size of Band/s Observed (bp) EcoRI 30, 15, 5 HindIII 50 EcoRI and HindIII 20, 15, 10, 5 a) How large (in base pairs) is this plasmid? There are 50 base pairs b) How many digests (cuts) does each restriction enzyme or combinations thereof make? EcoRI makes three cuts and HindIII makes one cut c) Use the above information to construct a restriction digest of the plasmid for these enzymes. Explain how you determined the positions of the restriction sites on your map. HindIII cut the plasmid once and EcoRI cuts three sites into fragments of 30, 15 and 5 bp. HindIII cuts one site at a fragment of 30bp, since the fragments 15bp and 5bp are present in the digest by the combination of the two resection enzymes. The fragment is divided into 20 and 10 base pairs and the result is shown below. Find a plasmid map of pBR322 and work out what would be the expected fragments sizes when this plasmid is cut by the following restriction enzymes: The pBR322 plasmid is shown below. Its total size is 4361bp a) EcoRI 4361 b) PvuII/EcoRI Pvull – 2064bp, 4359 – 2064 = 2295 4361 – 2295 = 2066 Pvull/EcoRI produce 2295bp, 2066bp c) EcoRI/SalI Sall – 651bp 4359 – 651 = 3708 4361 – 3708 = 653 Thus EcoRI/sall produces 3708bp, 653bp d) SalI/PvuII Sall = 651bp and Pvull = 2064bp 2064 – 651 = 1413 4361 – 1413 = 2948 Thus SalI/PvuII produces 2948bp, 1413bp e) EcoRI/SalI/PvuII EcoRI= 4359bp, Sall = 651bp and Pvull = 2064bp 4359 – 2064 = 2295 2064 – 651 = 1413 4361 – (2295 + 1413) = 653 Thus EcoRI/SalI/PvuII produce 2295bp, 1413bp and 653bp fragments sizes 2) A circular bacterial plasmid (pBP1) contains in the tetracycline-resistance gene (tetR)3. Human genomic DNA has been digested and inserted into tetR of the plasmid. This particular plasmid clone (number 15) contains the cloned gene. You perform restriction digests with HindIII and EcoRV. The gel results are shown above, with the control being plasmid with no insert. Band sizes are given in kilobases (kb). You can assume that all bands are from linear molecules. Answers to the questions below a) Why might it be useful to have a restriction site in the middle of an antibiotic resistance gene of a cloning vector such as a plasmid? The restriction site will ensure that there is a cut in the plasmid which is important in ligating an insert. Also, the resistance gene is inactivated by cloning into these restriction sites and the consequent sensitivity to the antibiotic is used as a screen for the recombinant plasmids which has the DNA insert (Hartl, & Ruvolo, 2012). b) How many fragments are produced if the pBP1 plasmid is cut with an enzyme that cuts twice? 2 fragments How many fragments would be produced if this was a linear piece of DNA? 3 fragments c) What does the HindIII site tell you? Why? The HindIII site can be cut with a HindIII restriction enzyme and can produce sticky ends as it has an overhanging reading frame. HindIII restriction site is in tetracycline-resistance gene (tetR). The HindIII site can be cut to produce two fragments with sizes 5 and 2.5kb. d) What three things does the EcoRV site tell you? Why? There are two restriction sites due to the generation of two bands on the gel. EcoRV site cut produce blunt ends because symmetrical DNA cut and it is palindromic restriction site. EcoRV cut the plasmid into two fragments with the fragments sizes of 3kb and 4.5kb There is no double band since the total size of the two bands adds to 7.5kb (Hartl, & Ruvolo, 2012). d) Draw two simple maps of this plasmid – one with and one without the insert, marking the possible restriction sites of HindIII and EcoRV. Also mark the sizes of regions in between. HindIII 5, 2.5 EcoR 4.5, 3 HindIII and EcoRV 3, 2, 1.5, 1 Maps of the plasmid – one without the insert and one with the insert 3) Use the data below to construct a circular restriction map on DNA. The map must indicate the positions of the restriction sites and distance between them. DNA Sizes of Fragments (bp) Size 7950 BglII 7950 EcoRI 7950 HpaI 7950 BglII and EcoRI 5416, 2534 BglII and HpaI 6632, 1318 EcoRI and HpaI 4098, 3852 The restriction map of the plasmid is shown below. 4) The figures below shows the DNA fragment with a newly discovered gene, Gene I. in a study you wish to subclone Gene I into a vector. The restriction enzyme EcoRI is used to digest the vector DNA and the target DNA. The EcoRI fragment containing the open reading frame of Gene I is ligated into the EcoRI site of the vector. a) How many orientations, relative to promoter X in the vector, could the insert fragment be in? The insert fragment can ligate into the vector in 2 possible orientations. The orientations are shown below. Correct orientation: Reverse orientation: b) The single restriction enzyme(s) which can used to determine the orientation, relative to promoter X. The fragment which will be released has been drawn below the map(s). BamHI or PstI or HindIII restriction enzymes can be used to determine the orientation because they cut the insert once. There relative sizes would indicate the orientation which they are in. fro example the fragment obtained with BamHI digest produce a shorter fragment in the correct orientation compared to the fragment obtained in the reverse orientation as shown below. Correct orientation: Reverse orientation: (Hartl, & Ruvolo, 2012) c) In some circumstances it is important that the insert is inserted in the orientation such that the direction of the open reading frames in Gene I is the same as the direction of transcription from promoter X. When you want to express some characteristics in the target DNA, it is important that orientation is correct. In order to express the gene in the correct orientation, the cloned sequence is inserted with the start codon near the promoter side. The RNA polymerase machinery moves along the DNA in the 5’→3’ direction of the coding strand and thus aids in the transcription of gene 1, starting at the promoter region as well as built the RNA transcript base in 5'→3' direction which is antiparallel to the DNA (Whittall, & Sutton, 2010). d) If you desire that the insert is in this orientation and that the entire gene was inserted, which enzymes would be used instead to subclone the fragment with Gene I in the vector? Explain why you selected these and not others. The two enzymes which may be used to subclone the fragment with the gene in the vector are EcoRI and PstI. The fragment generated by these enzymes contains the entire gene. They also produce directional cloning as the target and the vector are both cleaved with two enzymes, different sticky ends are produced. The sticky ends will ensure that the fragments are inserted in required orientation (Hartl, & Ruvolo, 2012). Conclusion This practical has provided an opportunity for me to study the effects of restriction enzymes in a plasmid DNA as one the important technique in molecular biology. I have learned how to construct maps using the cut fragments sizes which has enhanced my practical knowledge in the analysis of gene using mapping restriction. It has provided broader understanding of the application of the techniques in digestion of DNA using restriction enzymes, conditions of reaction and gel electrophoresis. The practical has also improved my confidence to analyze the gel data. This knowledge will very useful in future studies and research. References Harisha, S. (2007). Fundamentals of bioinformatics. New Delhi: I.K. International Publishing House. Hartl, D. L., & Ruvolo, M. (2012). Genetics: Analysis of genes and genomes. Burlington, MA: Jones & Bartlett Learning. Tropp, B. E. (2014). Principles of molecular biology. Burlington, MA: Jones & Bartlett Learning. Turner, P. C. (2000). Molecular biology. Oxford: BIOS. Whittall, J., & Sutton, P. (2010). Practical methods for biocatalysis and biotransformations. Chichester, U.K: J. Wiley. Meltzer, S. J. (1998). PCR in bioanalysis. Totowa, N.J: Humana Press. Read More