Extraction of Bacterial Chromosomal DNA - Essay Example

NUMBER:…………………………………………. SHS80204-2 Clinical Microbiology & Molecular Biology Practicals Practical Week 28th of January DNA extraction from bacteria Restriction of DNA Results and Discussion for Portfolio. To be handed in in-class on the 11th of February 2010. Extraction of bacterial chromosomal DNA Results. Micrococcus luteus For the diluted DNA OD260 = 0.017ul OD280 = 0.011ul . Dilution factor = 30 fold For the DNA OD260 = 0.51ul OD280 =0.33ul The ratio of OD260/OD280 = 1.545 Convert this to g/ml of DNA using the conversion 1.0 OD260 = 47 g/ml of DNA Concentration of isolated DNA =23.97ug/ml Total volume of DNA =200l. Yield of DNA = 4.794 g. This amount of DNA was isolated was isolated from 0.4 ml of a 5% w/v suspension. What was the dry weight of bacteria used? 0.4 / 100 x 5= 0.02 What % of the bacterial dry weight is DNA? 4.794/ 0.02= 239.7% Discussion/ Questions. 1. At which point in the protocol do you think the cells burst open? The bacterial cells burst open at the initial point in the protocol when the barriers like cytoplasic membrane and cell wall are disrupted by the enzyme lysozyme. 2. What is lysozyme and where is it usually found in animals? Lysozyme is an enzyme that digest the polymeric compounds that give the cell wall its rigidity. It is present in secretios like tears and saliva as well as in egg white. 3. What is SDS? SDS is a detergent Sodium Dodecyl Sulphate which aids in the disintegration of the bacterial cell wall. 4. What is the role of the following in the extraction process? Lysozyme Burst open cell wall by digesting the polymeric compounds that give cell wall its rigidity SDS Sodium Dodecyl Sulphate helps in cell wall lysis by removing lipid molecules sodium perchlorate Sodium perchlorate in higher concentrations will remove the detergent SDS and protein complexed with it and prevent the precipitation of proteins by ethanol during the concentration of DNA by ethanol precipitation. chloroform/isoamyl alcohol Chloroform/isoamyl alcohol are organic solvents which precipitate proteins but leave nucleic acids in aqueous solution which after centrifugation will result in distinct layers. Ethanol Etahnol is used to concentrate DNA by precipitation in the presence of monovalent cations like Sodium ions. 5. What is contained in the material at the interface of the organic and aqueous phases after centrifugation? Precipitated protein molecules are left as a white coagulated mass at the interface between the organic and aqueous layer. 6. Why is the DNA retained in the aqueous phase? DNA is retained in the aqueous phase as the chloroform/isamyl alcohol precipitates the non-polar substances like lipids and proteins leaving DNA in aqueous phase after centrifugation. 7. What % of the bacterial dry weight is DNA? 239.7%(according to calculation above) 8. Comment on the accuracy of this figure. The figure seems compatible with the data obtained from the practical. 9. From the optical densities and the ratios, is there any suggestion of contamination in the samples and why? If so, what do you imagine the contaminants are? The optical densities shows a slightly lesser absorbance than general for pure DNA samples indicating the presence contaminants in the sample. OD260/OD280 ratio is used as an estimate of DNA purity with a ratio between 1.7 and 2.0 accepted at high purity. The present sample shows a ratio of 1.54 indicating contamination. The contaminants could be RNA and other proteins which also shows a peak of absorbance at 260 nm. 10. Nucleic acids have a peak of absorbance at 260 nm and this is used to estimate their concentration. Why is the OD280 useful as a measure of contamination and what type of contamination is it measuring? OD260/OD280 ratio is used as an estimate of DNA purity with a ratio between 1.7 and 2.0 accepted at high purity. Aromatic amino acids present in proteins absorb at 280 nm which if present in the DNA sample will contribute to the total absorbance. 11). What modifications to the procedure do you imagine might have to be made to extract DNA from human cells? In order to extract DNA from human cells modifications are needed at the cell breakage stage as human cells have no cell wall as opposed to bacterial cells. Hence the cell can be lysed simply by treating it with a detergent instead of using lysozyme. 12). For what purposes might DNA extraction be relevant in a clinical setting - consider extraction from bacteria and also generally say from human tissue. DNA extraction in a clinical setting is relevant in a number of ways including PCR amplification for diagnosis of bacterial and other infection, methods like RFLP for locating genetic disorders etc. References 1. Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, Vol. 2, John Wiley and Sons, NY. 2. Birnboim, H.C. and Doly, J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513–23. 3. Brown, T.A. Gene Cloning and DNA Analysis An Introduction, 4th Edition Blackwell Publishing, 4. Glasel, J.A. (1997) Validity of nucleic acid purities monitored by 260nm/280nm absorbance ratios. BioTechniques 18, 62–3. 5. Lewin, B. (2004) In: Genes VIII Pearson Prentice Hall, Upper Saddle River, NJ 6. Promega Corporation (2002) Genomic DNA purification from blood. Application Notes 101, 1–4. 7. Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 8. Wilcockson, J. (1973) The use of sodium perchlorate in deproteinization during the preparation of nucleic acids.Biochem. J. 135, 559–61. Restriction of DNA Cutting DNA with restriction enzymes. 1). If the linear DNA molecule below is cut once () by the restirction enzyme how many fragments will be produced? Two DNA _________________________- 2). If there are two restriction sites (cutting sites) for the enzymes how many fragments will be produced? Three DNA _________________________ 3). What is the simple rule relating the number of fragments (these can be counted from an agarose gel - see next week) to the number of restriction sites present on the LINEAR DNA molecule? Number of fragments= Number of restriction sites+1 4). If the DNA molecule was circular, what would the rule be? Number of fragments= Number of restriction sites 5). When a linear DNA molecule was cut with EcoRI, the following sized fragments were produced,160 bp, 1500 bp, 2700 bp, 5600 bp. What was the total size of the original DNA and how many restriction sites were present for EcoRI? Total size= 9960 bp Restriction sites= 3 6). A circular DNA plasmid was cut with HindIII, the following sized fragments were produced,250bp, 600 bp, 5200 bp, 6000 bp. What was the total size of the original plasmid and how many restriction sites were present for HindIII? Total size = 12050 bp Restrition sites= 4 7). Go to the website called Webcutter at http://rna.lundberg.gu.se/cutter2/index.html This program site will cut a given piece of DNA with any enzyme you choose. Copy and Paste the following DNA sequence into the program and cut it with the following enzymes - attach the restriction map of the DNA - for each enzyme say how many sites there are, the position of the sites and the sizes of the fragments after cutting. Cut the DNA with HaeIII, EcoRI, HindIII. The DNA sequence is TATCTGAAGCGCAATCTTCGGACATTTTAGTATGGAACTTGACTCCTAGACAACTATGTG ATATTGAATTGATTCTAAATGGTGGGTTTTCTCCTCTGACTGGGTTTTTGAACGAAAACG ATTACTCCTCTGTTGTTACAGATTCGAGATTAGCAGACGGCACATTGTGGACCATCCCTA TTACATTAGATGTTGATGAAGCATTTGCTAACCAAATTAAACCAGACACAAGAATTGCCC TTTTCCAAGATGATGAAATTCCTATTGCTATACTTACTGTCCAGGATGTTTACAAGCCAA ACAAAACTATCGAAGCCGAAAAAGTCTTCAGAGGTGACCCAGAACATCCAGCCATTAGCT ATTTATTTAACGTTGCCGGTGATTATTACGTCGGCGGTTCTTTAGAAGCGATTCAATTAC CTCAACATTATGACTATCCAGGTTTGCGTAAGACACCTGCCCAACTAAGACTTGAATTCC AATCAAGACAATGGGACCGTGTCGTAGCTTTCCAAACTCGTAATCCAATGCATAGAGCCC ACAGGGAGTTGACTGTGAGAGCCGCCAGAGAAGCTAATGCTAAGGTGCTGATCCATCCAG TTGTTGGACTAACCAAACCAGGTGATATAGACCATCACACTCGTGTTCGTGTCTACCAGG AAATTATTAAGCGTTATCCTAATGGTATTGCTTTCTTATCCCTGTTGCCATTAGCAATGA GAATGAGTGGTGATAGAGAAGCCGTATGGCATGCTATTATTAGAAAGAATTATGGTGCCT CCCACTTCATTGTTGGTAGAGACCATGCGGGCCCAGGTAAGAACTCCAAGGGTGTTGATT TCTACGGTCCATACGATGCTCAAGAATTGGTCGAATCCTACAAGCATGAACTGGACATTG AAGTTGTTCCATTCAGAATGGTCACTTATTTGCCAGACGAAGACCGTTATGCTCCAATTG ATCAAATTGACACCACAAAGACGAGAACCTTGAACATTTCAGGTACAGAGTTGAGACGCC GTTTAAGAGTTGGTGGTGAGATTCCTGAATGGTTCTCATATCCTGAAGTGGTTAAAATCC TAAGAGAATCCAACCCACCAAGACCAAAACAAGGTTTTTCAATTGTTTTAGGTAATTCAT TAACCGTTTCTCGTGAGCAATTATCCATTGCTTTGTTGTCAACATTCTTGCAATTCGGTG GTGGCAGGTATTACAAGATCTTTGAACACAATAATAAGACAGAGTTACTATCTTTGATTC AAGATTTCATTGGTTCTGGTAGTGGACTAATTATTCCAAATCAATGGGAAGATGACAAGG ACTCTGTTGTTGGCAAGCAAAACGTTTACTTATTAGATACCTCAAGCTCAGCCGATATTC AGCTAGAGTCAGCGGATGAACCTATTTCACATATTGTACAAAAAGTTGTCCTATTCTTGG AAGACAATGGCTTTTTTGTATTTTAATCGTCATAAAATGCTCCCATCTCAAAAGTAGGGC AAAATTCATGATCGACCGCGCAAAATAAATAGATTTGCAAATAAGTTTTGTATGTACATT TATTAATATATATAATATATCAAAAGAAAAAAATCAAAAAAAAAAAAAAAAAAAAATTGC ACTCTTATTCAGTCATCAATTACAAAACCTAGAGATAGCGATGGTGCATATTCAATAAAA AACTCCTTATACTGTCGAGAAAGCTTATTATTGGTACTTCTCGAAGATACTAAAAAAGGT TAATTTTTGGAGACGGAGGCAATAGCGACTAGTCATACTGGTCGACATTAATTTCAATTT TTGGCTGTGCCCACCTCAATTTTTGTTTATTATACCATGGGTAAGCTGGCAAAACACATA TTAAAGTAATCACACAACTAATGCCATACGCAGTCAGTAAAACTTTCAGGGACTGTTGTG CAAATCCCAATATACACGCTACCAAAGCTCCGATCATGAGCGATAGCTGTTGAAATTTTT CTGTTTTCCGTTGGCTGGGAAAATCTATTGGAAAAACCAGTTTCCTTTGTACATCTTGTA ATATCTCAGACATTGAAGTTGACTATGACGTGACGTTGATCTTGTTTGAGTATTGCTTGA CCAATCCTGGAGTAACTTCGTTTAATCTGCCTCGTTTTTTTCTCTAAATAGTTTCTATTA TCCCTCCTCCACGCGAGATGTATTTAAAATGTGAAAAGTGTACACATGGCGTTCATCTTA CGTAGATGAGAGAAACGCTAAGAAGCCAACCAAACTAGTATAATCTATACATTTATAGAT GTTTGTGTAGAACCTTATTTTTATACTCTTGTATTATATTACATTACTTGCATTAAACCG TCATTTCACCAATAATTTGGTCCTTTAGCCAATTAACATATCTGATGAATGGTTCACCCT TGCTAACATCAATAACACTATCGTTCGTAGCTTTTCTATATATATTAATCAGGCCCTTAT CCTTAGAGAGCTCGTTAAATAGTTTACAATTCAAAAGATCTAACTGAAACTTCCAAATGG GTAACAATTCATTCTGTAACACATTAACTAAGATCTTCATCTGTAAATCGTTCATCTGTG ACTGTTGTTTACTCATAGGGATCTTGTTCAAAAATTTGTAGAGATTAACGAATCCTGATA AACAATTTTGCATTTGTAATATAAATTCATCTTTAAAATCGACATTTAATCTGTTTAATT CCTGGAAAATATACCTGTTCACTCCATAAATGGAGGTGACTAGAACCTTCTCGATCAAAT GCCGGCATTTTCCAGGAGTTTCAAAGTGATCTAACTCACAACTATCCAATAATAAGCGGA ATCCTGTCCTTAAATCGTAAATTAGTTCACAAATATCACTCTTTAGTGCAAGGAGTTCTT TTTTGTGTGAAATAAATGTGCCAGAATGGTTTAATACTTCGTCTTCCTGCTTTTGAGCCA GATAATACAAGACATACATTAGATCCGTCATATAATGTGAAGTATTCAGAATAAACGGCA CTATTTGATTCAGCAGTATAACATTTTTCTTGTTGTTTTCGGATATTCCGTCATTGAGCA AAAGTTTGGTTTGTAAAGACGAAACTGTACTTTCATTAGATTCTCGTGTTTCACTCATAC GCCTTTATATGCTATCACAGTTACCTTATTTGTCCTTTTTCTTGTAGAGCTATTCTCTAT TCTGCTTTGACTTAACTAGCGTGAAATTAAAACCAACGCCGCTTATCTAATCACAATAAA AGATCAGACTGTGAAATTAATAAAAAGGAAAGGATAAAACGACAGATATCGCACTAACTT TGTTAGAAACATTATGCAGACAAAAGAAGGAGATGCATGTGCCTTTGTATTCTTTTAAAC GCAAAAAGGGCAGGTCTATGATTAAGTATGAATGAGCGATATAAAATTTAAGTAGAAAAG GGAAGATAATAGTACTATATATAAATATATAAATGCATTTTTTTACATACCTGTTGGGTG AATAATTTACCTAGCTGTTGGATTTGCTGTTACTGTATTATTGTCGTCATCATTGCCATC GCCTTTATTGTTATCATTGTTATTGTCATCATTATCGTTGTCACTGTCATTAATCTGTAA CTTAGACTGTATATCGTAACATTCAGACAGCCTTGTTAATGAGGTCATGCCTTCTAGGCT TTCTTGATCAATGTCCAGAGATATATTGGGTTTGTCTAATGATCCTTGATGAGATTGATG CTGCAGCTGACCTTCCCAAGGCCTAGGTCTTGGCCTCAATGAAGGAGTACTAGCTAAACC CTCATCCAGCGTAACATTGGGCACCTCCACAATATTAGGTATAGGCTTGTTATTCAGAAT GTGATCCAGCAACTCTTCATAACTAAGTTCACCACCTGCTGCCATGCTTGACTCGTTATG TTCGCAATCAATTTTCAAGAGGTAAACCGAAACATGTGGAGCTCTTTTACAGGCTCCGAT AATAATAAAAGAAACTTTAATAGTTGGTAAGAACTTGACAA. Untitled sequence 4001 base pairs Graphic map | Table by enzyme name | Table by site position tatctgaagcgcaatcttcggacattttagtatggaacttgactcctagacaactatgtgatattgaattgattc base pairs atagacttcgcgttagaagcctgtaaaatcataccttgaactgaggatctgttgatacactataacttaactaag 1 to 75 taaatggtgggttttctcctctgactgggtttttgaacgaaaacgattactcctctgttgttacagattcgagat base pairs atttaccacccaaaagaggagactgacccaaaaacttgcttttgctaatgaggagacaacaatgtctaagctcta 76 to 150 tagcagacggcacattgtggaccatccctattacattagatgttgatgaagcatttgctaaccaaattaaaccag base pairs atcgtctgccgtgtaacacctggtagggataatgtaatctacaactacttcgtaaacgattggtttaatttggtc 151 to 225 acacaagaattgcccttttccaagatgatgaaattcctattgctatacttactgtccaggatgtttacaagccaa base pairs tgtgttcttaacgggaaaaggttctactactttaaggataacgatatgaatgacaggtcctacaaatgttcggtt 226 to 300 acaaaactatcgaagccgaaaaagtcttcagaggtgacccagaacatccagccattagctatttatttaacgttg base pairs tgttttgatagcttcggctttttcagaagtctccactgggtcttgtaggtcggtaatcgataaataaattgcaac 301 to 375 ccggtgattattacgtcggcggttctttagaagcgattcaattacctcaacattatgactatccaggtttgcgta base pairs ggccactaataatgcagccgccaagaaatcttcgctaagttaatggagttgtaatactgataggtccaaacgcat 376 to 450 EcoRI agacacctgcccaactaagacttgaattccaatcaagacaatgggaccgtgtcgtagctttccaaactcgtaatc base pairs tctgtggacgggttgattctgaacttaaggttagttctgttaccctggcacagcatcgaaaggtttgagcattag 451 to 525 caatgcatagagcccacagggagttgactgtgagagccgccagagaagctaatgctaaggtgctgatccatccag base pairs gttacgtatctcgggtgtccctcaactgacactctcggcggtctcttcgattacgattccacgactaggtaggtc 526 to 600 ttgttggactaaccaaaccaggtgatatagaccatcacactcgtgttcgtgtctaccaggaaattattaagcgtt base pairs aacaacctgattggtttggtccactatatctggtagtgtgagcacaagcacagatggtcctttaataattcgcaa 601 to 675 atcctaatggtattgctttcttatccctgttgccattagcaatgagaatgagtggtgatagagaagccgtatggc base pairs taggattaccataacgaaagaatagggacaacggtaatcgttactcttactcaccactatctcttcggcataccg 676 to 750 HaeIII atgctattattagaaagaattatggtgcctcccacttcattgttggtagagaccatgcgggcccaggtaagaact base pairs tacgataataatctttcttaataccacggagggtgaagtaacaaccatctctggtacgcccgggtccattcttga 751 to 825 ccaagggtgttgatttctacggtccatacgatgctcaagaattggtcgaatcctacaagcatgaactggacattg base pairs ggttcccacaactaaagatgccaggtatgctacgagttcttaaccagcttaggatgttcgtacttgacctgtaac 826 to 900 aagttgttccattcagaatggtcacttatttgccagacgaagaccgttatgctccaattgatcaaattgacacca base pairs ttcaacaaggtaagtcttaccagtgaataaacggtctgcttctggcaatacgaggttaactagtttaactgtggt 901 to 975 caaagacgagaaccttgaacatttcaggtacagagttgagacgccgtttaagagttggtggtgagattcctgaat base pairs gtttctgctcttggaacttgtaaagtccatgtctcaactctgcggcaaattctcaaccaccactctaaggactta 976 to 1050 ggttctcatatcctgaagtggttaaaatcctaagagaatccaacccaccaagaccaaaacaaggtttttcaattg base pairs ccaagagtataggacttcaccaattttaggattctcttaggttgggtggttctggttttgttccaaaaagttaac 1051 to 1125 ttttaggtaattcattaaccgtttctcgtgagcaattatccattgctttgttgtcaacattcttgcaattcggtg base pairs aaaatccattaagtaattggcaaagagcactcgttaataggtaacgaaacaacagttgtaagaacgttaagccac 1126 to 1200 gtggcaggtattacaagatctttgaacacaataataagacagagttactatctttgattcaagatttcattggtt base pairs caccgtccataatgttctagaaacttgtgttattattctgtctcaatgatagaaactaagttctaaagtaaccaa 1201 to 1275 ctggtagtggactaattattccaaatcaatgggaagatgacaaggactctgttgttggcaagcaaaacgtttact base pairs gaccatcacctgattaataaggtttagttacccttctactgttcctgagacaacaaccgttcgttttgcaaatga 1276 to 1350 tattagatacctcaagctcagccgatattcagctagagtcagcggatgaacctatttcacatattgtacaaaaag base pairs ataatctatggagttcgagtcggctataagtcgatctcagtcgcctacttggataaagtgtataacatgtttttc 1351 to 1425 ttgtcctattcttggaagacaatggcttttttgtattttaatcgtcataaaatgctcccatctcaaaagtagggc base pairs aacaggataagaaccttctgttaccgaaaaaacataaaattagcagtattttacgagggtagagttttcatcccg 1426 to 1500 aaaattcatgatcgaccgcgcaaaataaatagatttgcaaataagttttgtatgtacatttattaatatatataa base pairs ttttaagtactagctggcgcgttttatttatctaaacgtttattcaaaacatacatgtaaataattatatatatt 1501 to 1575 tatatcaaaagaaaaaaatcaaaaaaaaaaaaaaaaaaaaattgcactcttattcagtcatcaattacaaaacct base pairs atatagttttctttttttagtttttttttttttttttttttaacgtgagaataagtcagtagttaatgttttgga 1576 to 1650 HindIII agagatagcgatggtgcatattcaataaaaaactccttatactgtcgagaaagcttattattggtacttctcgaa base pairs tctctatcgctaccacgtataagttattttttgaggaatatgacagctctttcgaataataaccatgaagagctt 1651 to 1725 gatactaaaaaaggttaatttttggagacggaggcaatagcgactagtcatactggtcgacattaatttcaattt base pairs ctatgattttttccaattaaaaacctctgcctccgttatcgctgatcagtatgaccagctgtaattaaagttaaa 1726 to 1800 ttggctgtgcccacctcaatttttgtttattataccatgggtaagctggcaaaacacatattaaagtaatcacac base pairs aaccgacacgggtggagttaaaaacaaataatatggtacccattcgaccgttttgtgtataatttcattagtgtg 1801 to 1875 aactaatgccatacgcagtcagtaaaactttcagggactgttgtgcaaatcccaatatacacgctaccaaagctc base pairs ttgattacggtatgcgtcagtcattttgaaagtccctgacaacacgtttagggttatatgtgcgatggtttcgag 1876 to 1950 cgatcatgagcgatagctgttgaaatttttctgttttccgttggctgggaaaatctattggaaaaaccagtttcc base pairs gctagtactcgctatcgacaactttaaaaagacaaaaggcaaccgacccttttagataacctttttggtcaaagg 1951 to 2025 tttgtacatcttgtaatatctcagacattgaagttgactatgacgtgacgttgatcttgtttgagtattgcttga base pairs aaacatgtagaacattatagagtctgtaacttcaactgatactgcactgcaactagaacaaactcataacgaact 2026 to 2100 ccaatcctggagtaacttcgtttaatctgcctcgtttttttctctaaatagtttctattatccctcctccacgcg base pairs ggttaggacctcattgaagcaaattagacggagcaaaaaaagagatttatcaaagataatagggaggaggtgcgc 2101 to 2175 agatgtatttaaaatgtgaaaagtgtacacatggcgttcatcttacgtagatgagagaaacgctaagaagccaac base pairs tctacataaattttacacttttcacatgtgtaccgcaagtagaatgcatctactctctttgcgattcttcggttg 2176 to 2250 caaactagtataatctatacatttatagatgtttgtgtagaaccttatttttatactcttgtattatattacatt base pairs gtttgatcatattagatatgtaaatatctacaaacacatcttggaataaaaatatgagaacataatataatgtaa 2251 to 2325 acttgcattaaaccgtcatttcaccaataatttggtcctttagccaattaacatatctgatgaatggttcaccct base pairs tgaacgtaatttggcagtaaagtggttattaaaccaggaaatcggttaattgtatagactacttaccaagtggga 2326 to 2400 HaeIII tgctaacatcaataacactatcgttcgtagcttttctatatatattaatcaggcccttatccttagagagctcgt base pairs acgattgtagttattgtgatagcaagcatcgaaaagatatatataattagtccgggaataggaatctctcgagca 2401 to 2475 taaatagtttacaattcaaaagatctaactgaaacttccaaatgggtaacaattcattctgtaacacattaacta base pairs atttatcaaatgttaagttttctagattgactttgaaggtttacccattgttaagtaagacattgtgtaattgat 2476 to 2550 agatcttcatctgtaaatcgttcatctgtgactgttgtttactcatagggatcttgttcaaaaatttgtagagat base pairs tctagaagtagacatttagcaagtagacactgacaacaaatgagtatccctagaacaagtttttaaacatctcta 2551 to 2625 taacgaatcctgataaacaattttgcatttgtaatataaattcatctttaaaatcgacatttaatctgtttaatt base pairs attgcttaggactatttgttaaaacgtaaacattatatttaagtagaaattttagctgtaaattagacaaattaa 2626 to 2700 cctggaaaatatacctgttcactccataaatggaggtgactagaaccttctcgatcaaatgccggcattttccag base pairs ggaccttttatatggacaagtgaggtatttacctccactgatcttggaagagctagtttacggccgtaaaaggtc 2701 to 2775 gagtttcaaagtgatctaactcacaactatccaataataagcggaatcctgtccttaaatcgtaaattagttcac base pairs ctcaaagtttcactagattgagtgttgataggttattattcgccttaggacaggaatttagcatttaatcaagtg 2776 to 2850 aaatatcactctttagtgcaaggagttcttttttgtgtgaaataaatgtgccagaatggtttaatacttcgtctt base pairs tttatagtgagaaatcacgttcctcaagaaaaaacacactttatttacacggtcttaccaaattatgaagcagaa 2851 to 2925 cctgcttttgagccagataatacaagacatacattagatccgtcatataatgtgaagtattcagaataaacggca base pairs ggacgaaaactcggtctattatgttctgtatgtaatctaggcagtatattacacttcataagtcttatttgccgt 2926 to 3000 ctatttgattcagcagtataacatttttcttgttgttttcggatattccgtcattgagcaaaagtttggtttgta base pairs gataaactaagtcgtcatattgtaaaaagaacaacaaaagcctataaggcagtaactcgttttcaaaccaaacat 3001 to 3075 aagacgaaactgtactttcattagattctcgtgtttcactcatacgcctttatatgctatcacagttaccttatt base pairs ttctgctttgacatgaaagtaatctaagagcacaaagtgagtatgcggaaatatacgatagtgtcaatggaataa 3076 to 3150 tgtcctttttcttgtagagctattctctattctgctttgacttaactagcgtgaaattaaaaccaacgccgctta base pairs acaggaaaaagaacatctcgataagagataagacgaaactgaattgatcgcactttaattttggttgcggcgaat 3151 to 3225 tctaatcacaataaaagatcagactgtgaaattaataaaaaggaaaggataaaacgacagatatcgcactaactt base pairs agattagtgttattttctagtctgacactttaattatttttcctttcctattttgctgtctatagcgtgattgaa 3226 to 3300 tgttagaaacattatgcagacaaaagaaggagatgcatgtgcctttgtattcttttaaacgcaaaaagggcaggt base pairs acaatctttgtaatacgtctgttttcttcctctacgtacacggaaacataagaaaatttgcgtttttcccgtcca 3301 to 3375 ctatgattaagtatgaatgagcgatataaaatttaagtagaaaagggaagataatagtactatatataaatatat base pairs gatactaattcatacttactcgctatattttaaattcatcttttcccttctattatcatgatatatatttatata 3376 to 3450 aaatgcatttttttacatacctgttgggtgaataatttacctagctgttggatttgctgttactgtattattgtc base pairs tttacgtaaaaaaatgtatggacaacccacttattaaatggatcgacaacctaaacgacaatgacataataacag 3451 to 3525 gtcatcattgccatcgcctttattgttatcattgttattgtcatcattatcgttgtcactgtcattaatctgtaa base pairs cagtagtaacggtagcggaaataacaatagtaacaataacagtagtaatagcaacagtgacagtaattagacatt 3526 to 3600 cttagactgtatatcgtaacattcagacagccttgttaatgaggtcatgccttctaggctttcttgatcaatgtc base pairs gaatctgacatatagcattgtaagtctgtcggaacaattactccagtacggaagatccgaaagaactagttacag 3601 to 3675 HaeIII cagagatatattgggtttgtctaatgatccttgatgagattgatgctgcagctgaccttcccaaggcctaggtct base pairs gtctctatataacccaaacagattactaggaactactctaactacgacgtcgactggaagggttccggatccaga 3676 to 3750 HaeIII tggcctcaatgaaggagtactagctaaaccctcatccagcgtaacattgggcacctccacaatattaggtatagg base pairs accggagttacttcctcatgatcgatttgggagtaggtcgcattgtaacccgtggaggtgttataatccatatcc 3751 to 3825 cttgttattcagaatgtgatccagcaactcttcataactaagttcaccacctgctgccatgcttgactcgttatg base pairs gaacaataagtcttacactaggtcgttgagaagtattgattcaagtggtggacgacggtacgaactgagcaatac 3826 to 3900 ttcgcaatcaattttcaagaggtaaaccgaaacatgtggagctcttttacaggctccgataataataaaagaaac base pairs aagcgttagttaaaagttctccatttggctttgtacacctcgagaaaatgtccgaggctattattattttctttg 3901 to 3975 tttaatagttggtaagaacttgacaa base pairs aaattatcaaccattcttgaactgtt 3976 to 4001 Table by Enzyme Name Enzyme No. Positions Recognition name cuts of sites sequence EcoRI 1 474 g/aattc More info HaeIII 4 811 2453 3741 3753 gg/cc More info HindIII 1 1701 a/agctt More info Every enzyme analyzed cuts this sequence. Table by Site Position Cut Enzyme No. Positions Recognition site name cuts of other sites sequence 474 EcoRI 1 g/aattc More info 811 HaeIII 4 2453 3741 3753 gg/cc More info 1701 HindIII 1 a/agctt More info 2453 HaeIII 4 811 3741 3753 gg/cc More info 3741 HaeIII 4 811 2453 3753 gg/cc More info 3753 HaeIII 4 811 2453 3741 gg/cc More info 8). Look up the term RFLP- briefly what does it mean and how is it relevant to diagnosing genetic disease? Restriction fragment length polymorphism or RFLP refers to a difference in homologous DNA sequences that can be identified by the presence of fragments of different lengths after digestion of the concerned DNA samples with specific restriction edonucleases. RFLP is an important tool in diagnosing genetic diseases by localizing genes for genetic disorders. An RFLP probe is a labelled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they are separated by gel electrophoresis, revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. . References: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml Read More