TABLE 21.3 Some Restriction Enzymes Used in Gene Cloning Restriction Enzyme" Sequence Recognized' Bacterlal Source ВаmнI Bacillus amyloliquefaciens H 5'-GGATCC-3' 3'-CCTAGG-5' ↑ Sau3AI Staphylococcus aureus 3A 5'-GATC-3' 3'-CTAG-5' EcoRI Escherichia coli RY13 5'-GAATTC-3' 3'-CTTAAG-5' Nocardia aerocolonigenes 5'-GCCGGC-3' 3'-CGGCCG-5' Nael Pstl Providencia stuartii 5'-CTGCAG-3' 3'-GACGTC-5' ↑

HgaI recognizes a specific 5 bp sequence. How frequently would you expect a specific 5 bp sequence to be found in any genome, considering that there are 4 possibilities for each of the 5 nucleotides in the restriction site sequence?

How does that length compare to that of restriction enzymes? Implications?

Restriction endonucleases are bacterial enzymes that cleave duplex (double-stranded) DNA at specific nucleotide sequences. The mode of replication of the animal virus SV40 has been investigated by using restriction endonucleases that cleave SV40 DNA into a number of unique segments. Like most viruses, SV40 DNA is circular. The map positions of the 11 fragments produced by a pair of restriction endonucleases are shown on the next page. Immediately following a 5 or 10 minute pulse of radioactively labeled thymidine, labeled SV40 molecules that have completed replication during the pulse are isolated. These newly replicated DNA molecules are digested by the restriction endonucleases and the resulting fragments are analyzed for the relative amounts of pulse label they contain. The results are in the table below. Assume that at the time the label was added there was a random population of replicating SV40 DNA molecules in all possible stages of synthesis. From the information given below,…

A linear DNA fragment was produced by digestion with the restriction enzyme, Xba1. This fragment with XbaI(X) sites on both ends was then further digested with HindIII (H) and EcoRI (E).  Draw a restriction map of the linear fragment based on the gel electrophoresis results shown below.  X         H         Marker             E          H/E                           __2000bp __                    __1500b                         __1300bp        __             __        __1000bp                    __                         __700bp             ­­__        __500bp                         __400bp                         __300bp                      __                         __200bp          __        __                         __100bp

Now that you’ve isolated the gene and made lots of copies, you need to insert the gene into something you can manipulate and move into your lab strain of E. coli.  You have access to the following vectors, either pBR322 or pUC19 (look them up here to see a map https://www.neb.com/products/dna-plasmids-and-substrates Please note the restriction sites in BOLD appear only once in the plasmid).  You may use either vector.  What is a vector? Which vector did you choose? Explain why you made that choice. What enzyme(s) will you use to place your fragment into the vector? Explain why you chose these enzymes. How will you move this construct into coli? What phenotype will tell you if the coli have taken up the plasmid? What phenotype will tell you if the coli have taken up the plasmid with the gasP gene?