Read Vectors by Their Essential Parts
A cloning vector needs a way to replicate, a way to select, and suitable sites for inserting DNA.
Cloning Vectors is the central idea in this question pattern. Identify the condition in the stem before applying the method below.
Check the essentials
Vectors carry an origin of replication, selectable markers and useful restriction sites. Plasmids replicate independently of the chromosome. Insertional inactivation distinguishes recombinant from non-recombinant transformants; it does not identify cells that received no vector.
Know common vectors
pBR₃₂₂ carries ampicillin- and tetracycline-resistance markers; pUC₁₈ carries ampicillin resistance and lacZ. BACs and YACs accept large DNA inserts. YEp is a yeast–E. coli shuttle vector, while YAC means yeast artificial chromosome.
Origins of replication
A plasmid origin of replication is the sequence where DNA replication begins. It allows a recombinant plasmid to be copied independently in a bacterial host; antibiotic resistance is a separate selectable marker.
Use marker disruption to identify recombinants
BamHI cuts pBR₃₂₂ within tetR. Inserting foreign DNA there disrupts tetracycline resistance but leaves ampicillin resistance intact. Recombinant bacteria therefore grow on ampicillin and fail on tetracycline. First locate the cut site, then predict which marker is lost. Choose D.
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- ✓Vector: ori + marker + insertion sites.
- ✓pBR₃₂₂: ampR and tetR; pUC₁₈: ampR and lacZ.
- ✓lacZ disruption → white colonies in the common screen.
- ✓BAC/YAC → large inserts; YEp → shuttle vector.
- ✓Insertion into pBR₃₂₂ tetR gives ampR tetS recombinants.
Separate non-transformants (no vector) from recombinants (vector with an insert).
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