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Based on his _________, Redi made a prediction that keeping flies away from meat would prevent the appearance of maggots.

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*10.12 A colleague has sent you a 4,500-bp DNA fragment excised from a plasmid cloning vector with the enzymes PstI and BglII (see Table 8.1, p. 174, for a descriptionof these enzymes and the sites they recognize). Your colleague tells you that within the fragment there is an EcoRI site that lies 490 bp from the PstI site.a. List the steps you would take to clone the PstI-BglIIDNA fragment into the plasmid vector pBluescript II (described in Figure 8.4, p. 176).b. How would you verify that you have cloned the correct fragment and how would you determine its orientation within the pBluescript II cloning vector?

10.8 Some thermostable DNA polymerases used in PCR leave an unpaired A nucleotide at the ends of the amplified fragments. How can this be useful to clone the PCRproducts?

*10.22 The investigators described in Question 10.14 were successful in purifying a BAC clone containing the gene for the mouse prion protein. To narrow down whichregion of the BAC DNA contains the gene for the prion protein gene, they purified the BAC DNA, digested it with the restriction enzyme NotI, and separated the products of the enzymatic digestion by size using gel electrophoresis. Then they purified each of the relatively large NotI DNA fragments from the gel, digested each individually with the restriction enzyme BamHI, and separated the products of each enzymatic digestion by size using gel electrophoresis (see Table 8.1, p. 174, for a description of the sites recognized by NotI and BamHI). Finally, they transferred the size-separated DNA fragmentsfrom the agarose gel onto a membrane filter using the Southern blot technique, and allowed the DNA fragmentson the filter to hybridize with a labeled cDNA probe. Figure 10.C shows the results that were obtained: The pattern of DNA bands seen after the BAC DNA is digested with NotI is shown in Panel A, the pattern of DNAbands seen after each NotI fragment is digested with BamHI is shown in Panel B, and the pattern of hybridizing DNA fragments visible after probing the Southernblot is shown in Panel C.a. Note the scales (in kb) on the left of each figure. Whyare relatively larger DNA fragments obtained withNotI than with BamHI?b. An alternative approach to identify the BamHI fragmentscontaining the prion-protein gene would be todigest the BAC DNA directly with BamHI, separatethe products by size using gel electrophoresis, make aSouthern blot, and probe it with the labeled cDNAclone. Why might the researchers have added the additionalstep of first purifying individual large NotIfragments, and then separately digesting each withBamHI before making the Southern blot?c. Which NotI DNA fragment contains the gene for the mouse prion protein?d. Which BamHI fragments contain the gene for the mouse prion protein?e. About what size is the RNA-coding region of the genefor the mouse prion protein? Why is it so much largerthan the cDNA?

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