pob5e_solutions_ch09

pob5e_solutions_ch09 - 2608T_ch09sm_S99-S111 2/21/08...

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S-99 DNA-Based Information Technologies 1. Cloning When joining two or more DNA fragments, a researcher can adjust the sequence at the junction in a variety of subtle ways, as seen in the following exercises. (a) Draw the structure of each end of a linear DNA fragment produced by an Eco RI restriction digest (include those sequences remaining from the Eco RI recognition sequence). (b) Draw the structure resulting from the reaction of this end sequence with DNA polymerase I and the four deoxynucleoside triphosphates (see Fig. 8–33). (c) Draw the sequence produced at the junction that arises if two ends with the structure derived in (b) are ligated (see Fig. 25–17). (d) Draw the structure produced if the structure derived in (a) is treated with a nuclease that degrades only single-stranded DNA. (e) Draw the sequence of the junction produced if an end with structure (b) is ligated to an end with structure (d). (f) Draw the structure of the end of a linear DNA fragment that was produced by a Pvu II restriction digest (include those sequences remaining from the Pvu II recognition sequence). (g) Draw the sequence of the junction produced if an end with structure (b) is ligated to an end with structure (f). (h) Suppose you can synthesize a short duplex DNA fragment with any sequence you desire. With this synthetic fragment and the procedures described in (a) through (g), design a protocol that would remove an Eco RI restriction site from a DNA molecule and incorporate a new Bam HI restriction site at approximately the same location. (See Fig. 9–2.) (i) Design four different short synthetic double-stranded DNA fragments that would permit ligation of structure (a) with a DNA fragment produced by a Pst I restriction digest. In one of these fragments, design the sequence so that the final junction contains the recognition sequences for both Eco RI and Pst I. In the second and third fragments, design the sequence so that the junction contains only the Eco RI and only the Pst I recognition sequence, respectively. Design the sequence of the fourth fragment so that neither the Eco RI nor the Pst I sequence appears in the junction. Answer Type II restriction enzymes cleave double-stranded DNA within recognition sequences to create either blunt-ended or sticky-ended fragments. Blunt-ended DNA frag- ments can be joined by the action of T4 DNA ligase. Sticky-ended DNA fragments can be joined by either E. coli or T4 DNA ligases, provided that the sticky ends are complementary. Sticky-ended fragments without complementary ends can be joined only after the ends are made blunt, either by exonucleases or by E. coli DNA polymerase I. (a) The recognition sequence for Eco RI is (5 ± )GAATTC(3 ± ), with the cleavage site between G and A (see Table 9–2). Thus, digestion of a DNA molecule with one Eco RI site chapter 9 2608T_ch09sm_S99-S111 2/21/08 11:45AM Page S-99 ntt Os9:Desktop Folder:TEMPWORK:FEBRUARY:21-02-08:WHQY028/soln:
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S-100 Chapter 9 DNA-Based Information Technologies (5 ± ) , GAATTC , (3 ± ) (3 ± ) , CTTAAG , (5 ± ) would yield two fragments: (5 ± ) , G(3 ± ) and (5 ± )AATTC , (3 ± ) (3 ± ) ,
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