How is DNA Manipulated? 1. Explain the following terms and their role in recombinant DNA technology a) Restriction enzyme ___cuts DNA into fragments_____ b) Recognition site ___specific site or spot where the enzyme cuts__ c) Sticky end ______area of DNA where bases are ready to be paired, they will "stick" to matching DNA__ d) Ligation _____________the joining of two different DNA________________ e) rDNA ________the new DNA is called DNA, or recombinant DNA_____________ Here are some restriction enzymes and their sites. G/GATCC PstI CTGCA/G HindIII A/AGCTT HhaI GCG/C EcoRI G/AATTC HpaII C/CGG SalI G/TCGAC HindII GTC/GAC BAMHI CAG/CTG 2. On each of the sequences below, determine which restriction enzyme could be used to splice the DNA and indicate where the cut will be made and the enzyme used. 1. 5' T T T G / A A T T C A G A T 3' 3' A A A C T T A A / G T C T A 5' 2. 3. Enzyme: 3' C A C C C T A G / G G A A T 5' _ 5' A C G C C T C / C G G A G A 3' 5' T T A / A G C T T A A G A A G C T T 3' 3' A A T T C G A / A T T C T T C G A A 5' 5. ___EcoRI____ 5' G T G G / G A T C C C T T A 3' 3' T G C G G A G G C / C T C T 5' 4. Enzyme: 5' A A G C G / C G T C G A C T T A T A 3' 3' T T C / G C G C A G C T G A A T A T 5' ___BAMHI_______ Enzyme: ____HpaII____ Enzyme: __HindIII________ Enzyme: ____HhaI________ 3. Create a restriction enzyme that will remove the gene of interst. Give it a name too! 4. The following DNA sequence is from a virus that is dangerous, scientists want to use a restriction enzyme to cut the virus into bits. They do not need sticky ends because the do not plan to combine it with other DNA. Use HindII to show how this DNA would be cut. How many pieces would you have? __4__ G A A A A G T C / G A C A A G G C A G T C / G A C T T T T A A A A G T C / G A C A T G C C T T T T C A G / C T G T T C C G T C A G / C T G A A A A T T T T C A G / C T G T A C G How is the Recombinant DNA (rDNA) Used? a) Which genes are being isolated from the flu virus? ____HA and NA_____ b) Where are the flu genes (HA and NA) placed? ____plasmids___________ c) Where do plasmids come from and why are they imporant in this process? _they copies of the gene___ d) What are the potential applications for making a new flu? ______developing vaccines___________ make