01/162.101 NIALB rnal only CP ASR TR NSB Sc ESTION 10 EcoRI BamHl Accl Hindil-1

Question

01/162.101 NIALB rnal only CP ASR TR NSB Sc ESTION 10 EcoRI BamHl Accl Hindil-1 Ori PONE AmpR TetR BamHl Suppose you have a human gene with Accl, BamHI, EcoRI and Hindil restriction enzyme recognition sites on both sides of the gene. You want to insert your gene into the plasmid, pONE (shown above), which contains two selectable markers, TetR and AmpR, as well as the restriction enzyme sites shown. For each of the restriction enzyme(s), say whether it would be a good choice to use or not (circle yes or no), and explain your answers. (6 marks) yes /no because: (ii) BamHI: yes /no because (ii) EcoRI: yes /no because (iv) Hindl: yes / no because:

Explanation / Answer

Answer:

Say whether it would be a good choice to use or not use and explain your answers:

Sno. RE Yes/No Because 1 AccI No AccI doesnot has more than 1 recognition site in the given pONE plasmid. Presence of duplicates of the restriction site anywhere in the gene insert or vector will cause multiple cuts in the given DNA and can lead to erroneous or misleading data. 2 BamHI No Recognition site for BamHI is present in the selectable marker genes for Tetracyclin and Ampicillin Resistance. Any insertion in these sites will affect the selection of transformed colonies as it will render transformed colonies susceptible and not resistant to antibiotics. 3 EcoRI Yes Yes, EcoRI has a single recognition site in the given plasmid and it can be used for inserting gene 4 HindIII No No, because HindIII recognition site is present in Ori (origin of replication). Ori is the DNA sequence that signals for the origin of replication and its the region where the DNA replication machinery assembles.

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