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 EcoRI restriction digest (include those sequences remaining from the EcoRI recognition sequence). (b) Draw the structure resulting from the reaction of this end sequence with DNA polymerase I and the four deoxynucleoside triphosphates. (c) Draw the sequence produced at the junction that arises if two ends with the structure derived in (b) are ligated
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 EcoRI restriction digest (include those sequences remaining from the EcoRI recognition sequence).
(b) Draw the structure resulting from the reaction of this end sequence with DNA polymerase I and the four deoxynucleoside triphosphates.
(c) Draw the sequence produced at the junction that arises if two ends with the structure derived in (b) are ligated
(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 PvuII restriction digest (include those sequences remaining from the PvuII 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 EcoRI restriction site from a DNA molecule and incorporate a new BamHI restriction site at approximately the same location.
(i) Design four different short synthetic double-stranded DNA fragments that would permit ligation of structure (a) with a DNA fragment produced by a PstI restriction digest. In one of these fragments, design the sequence so that the final junction contains the recognition sequences for both EcoRI and PstI. In the second and third fragments, design the sequence so that the junction contains only the EcoRI and only the PstI recognition
sequence, respectively. Design the sequence of the fourth fragment so that neither the EcoRI nor the PstI sequence appears in the junction.
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