During your study of a biologically active sequence, you decided to develop a se
ID: 164174 • Letter: D
Question
During your study of a biologically active sequence, you decided to develop a series of mutations on your peptide sequence to probe sequence versus activity profiles. You develop a DNA mutation experiment, the following plasmid (entire not shown just the site of interest) and DNA insert sequence was worked with. Answer the following questions using these sequences. pts) Plasmid Sequence. ACGTTCACAGCTGGGTACCGGATGGCATGCCGAGTTTGTCGACTGAGGGTACCGTCGAGGGACC TGCAAGTGTCGACCCATGGCCTACCGTACGCTCAAACAGCTGACTCCCATGGCAGCTCCCTG recognize a plasmid is typically circular. For restriction endonucleases do not cleave any where else in the sequence s this exercise, assume that outside of this region. DNA Insert Sequence ATTACGGCAGCTGGGTACCATATGGCTTGTGAATTCGTAGATTAATGGTACCGTCGAGC TTAC -TAATGCCGTCGACCCATGGTATACCGAACACTTAAGCATCTAATTACCATGGCAGCTCGAATG During the first step of the plasmid mutation experiment, you need to use a restriction endonuclease. Which one of the following listed would you suggest Explain why. Sphl EcoRI Kpnl Povll Sall upon insertion of the new DNA sequence into the plasmid, you synthesize your "mutant" heptapeptide and compare it to the original sequence (the However, you find that nothing has changes between the mutant and wild-type. Therefore, you go back and make sure you have done everything you set out to do in the experiment. As part of this check-up, determine the amino acid for each the (from start to stop codon). sequences above have been provided below for this exercise. Wild-type Sequence ACGTTCACAGCTGGG -TGCAAGTGTCGACCCATGGCCTACCGTACGCTCAAACAGCTGACTcGCATGGCAGO TCCCTGG Mutant sequence ATTACGGCAGCTGGGTACCATATGG CTTGTGAATTCGTAGATTAATAGTACCGTCGAGCCTTAC GCCGTCGACCCATGGTATACCGAACACGCTCGAATGExplanation / Answer
In this context where a polypeptide contains a heptapeptide is biologically active sequence which is to form a mutant version of it with a DNA insert and a Plasmid part. To make a mutant version we need to use a restriction digestion enzyme, from that we know restriction endonicleases (RE) precisely make a cut in a DNA sequence internally from the portion of 5' also same site at the other end, from 3'. These endonucleases as name suggests are very particular and very useful to make a specific cut near specific recognition site that would help other DNA portion to join it by making the same cut in the joining end. RE's are generally 4 different types, out of that type II is most famous and widely used. It has got many variety and we generally name them from where or which microorganism we have found it.
From the given examples we can get to know about the specific cut sites of these RE's, then we can predict which is to be used in this mutation experiment.
From the given RE's we have choose KpnI, which has a restriction site G G T A C C, and we can see this sequence in both the plasmid as well as in the DNA insert, so from the restriction cut we can join the sequences. This is the perfect amtch for making the mutant insert possible, thus the correct RE is KpnI.
b) 21 upstream from the 5' sequence in the plasmid has got the start codon in the plasmid DNA as ATG (In wild type), so it is obvious that mutant version has got the same place to keep its start codon. After the hexapeptide plasmid wild type has got the TGA stop codon, also in the case of mutant version we can encounter the hexapeptide and then stop codon, here it is TAA.
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