MT2hardQanswers - Q The figure at left below shows a...

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Q. The figure at left below shows a eukaryotic gene with two exons, which is transcribed from left to right. The figure at right shows a plasmid that contains a bacterial promoter (shaded box) next to a multiple cloning site, so that a bacterial RNA polymerase can be used to transcribe any inserted DNA. You want to use this plasmid to make a labelled RNA probe that can be hybridized to transcripts from the gene, on Northern blots and in tissues. You plan to start by (i) cutting a restriction fragment from the gene at two of the enzyme sites shown; (ii) cutting the plasmid with the same two enzymes and (iii) ligating the fragment into the plasmid. Which gene fragment should you insert into the plasmid? Note that fragment ends cut with any of these enzymes can only be joined to other ends cut with the same enzyme – e.g., Eco-cut ends can only be joined to other Eco-cut ends. Answer : fragment 4 . To be able to hybridize to transcripts on an Northern blot or in tissues, the single- stranded RNA probe you are making must include sequences complementary to the gene mRNA. Fragments 1 (upstream DNA ) and 3 (intron) will not be in the processed transcripts. Fragments 2 and 5 do include exon sequence, but both, when inserted into the plasmid, will be transcribed to give a sense strand relative to the gene, due to the position of the restriction sites relative to the plasmid promoter.
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