CBMS 336: Molecular Biology and Genomics - DNA Cloning and Sequencing - Bioinformatics Assessment Answer

Subject Code: CBMS 336 Internal Code: E_AI_DGFJ_HB

Bioinformatics Assessment Answer

TASK: 1. Genomic DNA from a particular organism has a G/C content of 64%. What is the average DNA fragment length expected after digestion with the following restriction endonucleases. a) BamH1 GGATCC b) Dra1 TTTAAA c) DraIII CACNNNGTG d) BanII GRGCYC e) SfiI GGCCNNNNNGGCC Note: R = Purine Y = Pyrimidine 2. Explain the following. Restriction enzyme 1 acts at all sites acted on by restriction enzyme 2 to give a total of 80 fragments, however restriction enzyme 2 acts on only 5 of the sites acted on by restriction enzyme 1. 3. XhoII has the recognition site R/GATCY. BamHI has the recognition site G/GATCC. What fraction of BamHI sites will be cut by XhoII? What fraction of XhoII sites will be cut by BamHI? What fraction of XhoII cut DNA fragments will be ligatable into a BamHI cut vector? 4. ApaI has the recognition sequence GGGCC/C. BanII has the recognition sequence GRGCY/C. If you had a vector with an ApaI cloning site, what fraction of BanII generated restriction fragments could be successfully cloned into this vector? 5. 0.2pmoles of pUC18 DNA (2.7kb) is added to 150?l of competent cells. After heat hock, 1.0ml of L-broth is added to the cells and they are incubated for 1hr at 37 0 C. An aliquot of these cells is diluted 100 fold and 200?l of these cells are spread out on an agar plate supplemented with the antibiotic. The next day 350 colonies appear on the plate. How many transformants are there per ?g of DNA. 6. You have cut 100ng of pBR322 with BamH1. Into this you want to clone Sau3A fragments of average length 300 base pairs. How much insert DNA would you add to obtain a threefold molar excess over vector in your ligation? Under these conditions what is the Molarity of vector if the ligation is done in 30?l? 7. A protocol recommends that for efficient ligation you add 20 picomoles of a particular DNA to carry out the reaction. If your DNA is 420 base pairs long, how much of this DNA would you add to the ligation mix? 8. 1?g of lambda DNA is digested with HindIII. This is then electrophoresed. How much DNA (by weight) is present in the 23.1 kb band? How much DNA (by weight) is present in the 564 bp band? How many moles of DNA are present in each of these bands? 9. A particular sequence containing 6 base pairs is located in 10 different organisms. The observed sequences are ACGCAC, ATACAC, GTGCAC, ACGCAC, ATACAC, ATGTAT, ATGCGC, ACGCAT, GTGCAT and ATGCGC. What is the consensus sequence? 10. You have partially sequenced the protein from a kangaroo. The sequence is Leu Met Asp Cys Trp Ile Thr Phe Ile. You want to extract the corresponding gene from wombats which you believe has the same amino acid sequence as shown. To identify the gene you want to synthesise an oligonucleotide of 15 bases for use as a probe. What region of the peptide would you use to develop your oligo using the least number of combinations but giving a sequence that must be absolutely complementary to your corresponding wombat sequence? 11. Why do you think thymine is used in DNA and uracil used in RNA when the other three bases are the same in each? 12. Plasmid DNA is digested with a number of restriction enzymes for the purpose of determining a restriction map. Fragment lengths as measured off an agarose gel are as follows: BamH1: 4.7, 2.6 EcoR1: 12.0 Pst1: 10.2, 1.8 HindIII: 7.8, 4.2 Bam + Eco: 4.7, 2.6, 2.4, 2.3 Pst + Eco: 5.9, 4.3, 1.8 Pst + Hind: 7.8, 1.8, 1.4, 1.0 Bam + Pst: 4.7, 2.6, 1.9, 1.8, 1.0 Eco + Hind: 4.5, 4.2, 3.3 All sizes are in kb Construct a restriction map for this plasmid. Subject Code: CBMS 336

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