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10.7 One frequent objective of expressing a protein in E. coli using an expression vector is to purify it. If the expressed protein is "tagged" at one end with a particular peptide sequence, it is easier to purify. For example, proteins tagged at one end with six histidine residues can berecovered from lysed E. coli cells by incubating the lysate with a nickel-containing resin. The six-histidine tag has a high affinity for the resin, facilitating the purification of the protein from the lysate. Proteins tagged in this way are fusion proteins—they contain the amino acid sequence encoded by an open reading frame (ORF) of a cDNA fused to the amino acid sequence of the tag. Some plasmid vectors have been designed to facilitate the production of such fusion proteins. They have an E. coli promoter sequence for transcription initiation and are designed so that the RNA that is produced will have a Shine-Dalgarno sequence near its5' end to facilitate ribosome binding.Following this, they have an ORF with the codons for the tag at its end. A multiple cloning site (MCS) is embedded within the ORF to facilitate cloning of part of a cDNA.Figure 10.B shows the MCS in one such vector. In the figure, the 5'-to-3' DNA strand that has the same polarity as the resulting mRNA is in bold type, the amino acids it encodes are given underneath their codons, and three unique restriction enzyme sites in the vector are shadowed in grey with the sites of DNA cleavage indicated by lines.a. Suppose your want to tag a polypeptide encoded by acloned cDNA whose ORF includes XhoI and EcoRI sites close to its beginning and a PstI site close to its end. What steps would you take to insert a fragment containing most of the cDNA's ORF into this expression vector? Can you be certain that a fusion protein will be produced?b. How would you clone the ORF of a previously cloned cDNA into this expression vector if the cDNA had no XhoI, PstI, or EcoRI sites? What concerns would you need to address to ensure that a fusion protein would be produced?

*10.43 As described in the text and demonstrated in Question 10.42, VNTRs can robustly distinguish between different individuals. Five well-chosen, single locus VNTR probes used together can almost uniquelyidentify one individual because, statistically, they are able to discriminate 1 in 10^9 individuals. However, the use of VNTR markers has largely been supplanted by theuse of STR markers. For example, the FBI uses a set of 13 STR markers in forensic analyses. Different fluorescently labeled primers and reaction conditions have been developed so that this marker set can be multiplexed—allof the markers can be amplified in one PCR reaction. The marker set used by the FBI, the number of alleles at each marker, and the probability of obtaining a randommatch of a marker in Caucasians is listed in the followingtable: Table Herea. Consider the types of DNA samples that the FBI analyzesand the requirements concerning DNA samples in the methods used to analyze STR and VNTR markers.Why is the use of STR markers preferable to the use of VNTR markers?b. Why is it advantageous to be able to multiplex the PCR reactions used in forensic STR analyses?c. Suppose the first four STR markers listed in the table are used to characterize the genotype of an individual, and the genotype is an exact match with results obtained from a hair sample found at a crime scene. What is the probability that the individual has been misidentified, that is, what is the chance of a random match when just these four markers are used? About how often do you expect an individual to be misidentified if only these four markers are used?d. Answer the questions posed in (c) if all 13 STR markersare used.

10.50 The ability to place cloned genes into plants raises the possibility of engineering new, better strains of crops such as wheat, maize, and squash. It is possible to identify useful genes, isolate them by cloning, and insertthem directly into a plant host. Usually these genes bring out desired traits that allow the crops in question to flourish. Why then is there such concern by consumersabout this process? Do you feel that the concern is justified?Defend your answer.

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