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Questions and Answers
What is a primary property of a cloning vector?
Cloning vectors must have the possibility of gene escape.
False
What is a selectable marker in cloning vectors?
A gene that confers resistance to particular antibiotics.
Which plasmid is used for in vitro cloning of eukaryotic DNA?
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PBR322 is a cloning vector that replicates in __________.
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What happens to the tet resistance gene when foreign DNA is inserted into the BamHI site of pBR322?
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Which of the following vectors is ideal for sequencing?
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Match the following plasmids to their properties:
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Study Notes
Cloning Vectors Overview
- Cloning vectors are small DNA pieces, often from viruses or plasmids, used to incorporate foreign DNA for cloning purposes.
Key Properties of Cloning Vectors
- Autonomous Replication: Vectors can replicate independently within a host cell.
- Small Size: Increases chances of unique restriction enzyme (RE) sites and enhances gene transfer efficiency.
- Selectable Marker Genes: Facilitate easy identification of recombinant DNA, commonly via antibiotic resistance.
- Multiple Cloning Sites: Allow insertion of DNA segments that bring about phenotypic changes, like loss of gene expression.
- Purification Ease: Vectors can be easily purified after cloning.
- Unaffected Replication: Insertion of foreign DNA does not hinder the vector's replication ability.
- High Efficiency Reintroduction: Vectors can be efficiently introduced back into host cells.
- Biological Containment: Prevents gene escape, achieved via non-conjugative plasmid vectors.
- Dual Origins of Replication: Shuttle vectors feature two different origins, enhancing compatibility between different host cells.
- Presence of Promoters/Ribosome Binding Sites: Essential for driving gene expression.
Common Features of Vectors
- Origin of Replication (ORI): Determines the vector's ability to replicate.
- Selectable Marker: Often antibiotic resistance, enhancing identification of successful cloning.
- Multiple Cloning Sites: Contain numerous restriction sites for flexible cloning options.
- High Copy Number: Ensures ample quantities of the vector for experiments.
Cloning and Selectable Markers
- Cloning Site: Specific location on the vector for inserting DNA; recombinant plasmids may feature up to 20 restriction sites.
- Selectable Markers: Genes that confer antibiotic resistance, allowing survival and growth in selective media.
- Reporter or Marker Genes: Aid in identifying successful clones, commonly through blue-white selection.
Additional Properties of Vectors
- Short and small for efficient manipulation.
- Must be compatible with the host cell, but incompatible with other vectors.
- High copy number to ensure sufficient production.
- Must utilize the host's cellular machinery for expression.
- Capable of replicating under two biological systems.
Types of Natural Plasmids
- pSC101: 9 Kbp, low copy number, single Eco RI site, provides tetracycline resistance, derived from R6-5 plasmid.
- Col E1 Plasmid: 6466 bp, circular, has genes for colicin production and immunity, high copy number, enhanced by chloramphenicol.
- pSF2124: Contains ampicillin resistance gene, capable of colicin biosynthesis, high copy number with unique sites for Bam HI and Eco RI.
Main Classes of Vectors
- Plasmid: Example: pUC.
- Viral: Examples: M13, Lambda.
Specific Plasmid Examples
- pBR322: Cloning vector for E. coli, features one ORI, single cleavage sites for various restriction enzymes, includes tet and amp resistance genes.
- pUC Series: Next-generation vectors, designed with a comprehensive multiple cloning site, efficient screening using X-gal.
- pGEM3Z: Lambda-based vector, able to undergo transduction and conjugation, promotes RNA expression.
Viral Vectors
- M13-Based Vector: Ideal for sequencing and phage display applications.
- Lambda-Based Vector: Capable of carrying large DNA segments and in vitro packaging.
Phage DNA Mechanism
- Phage employs a linear double-stranded DNA mechanism involving:
- Attachment to bacterial cells
- Insertion and replication within bacteria
- Utilizing bacterial machinery for its reproduction
- Bursting to release new phage particles, with COS site aiding circular DNA formation.
Additional M13 Characteristics
- Single-stranded nature, capable of conjugation using pili.
- Converts single-stranded DNA into double-stranded DNA for replication while slowing phage lysis.
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Description
Test your knowledge about cloning vectors, including their primary properties, selectable markers, and specific plasmids like pBR322. This quiz covers key concepts important for understanding molecular cloning techniques and their applications in genetics.
