Artificial Chromosome Vectors Overview

Artificial Chromosome Vectors

• DNA molecules assembled in vitro
• Used to clone large DNA fragments
• Used for genome sequencing

Characteristics

• ORI
• MCS
• Selectable Marker

Types

• BAC (Bacterial Artificial Chromosome)
  • Engineered DNA molecules used to clone DNA in bacterial cells
  • Primarily used for DNA fragment sizing 150-350 kb
  • Can handle large DNA inserts (1-3 million bp)
  • Used to study inherited diseases
  • Transferred to bacteria using gene gun or electroporation
• Components:
  • ORI (origin of replication)
  • RepE (regulation of plasmid copy number)
  • ParA & ParB (equal partitioning of plasmid DNA into daughter cells)
  • Selectable markers (antibiotic resistance genes)
  • Sp6 & T7 promoters (for transcription of inserted genes)
• YAC (Yeast Artificial Chromosome):
  • Shuttle vectors that can replicate in both E. coli and yeast
  • Used for cloning large DNA inserts (hundreds of kb to several Mb)
  • Components:
    • Centromere
    • Telomeres
    • ARS (autonomous replication sequence)
• Advantages of BAC over YAC:
  • Better stability
  • Lesser chance of chimerism (mixture of DNA)
• Applications:
  • Study herpesviruses
  • Study new viruses
  • Study cancer research models
  • Study genetic disorders
  • Study infectious diseases (viruses)

Lambda Vector

• Carrying capacity: 10 kb
• Temperate phage that infects E. coli
• Replicates by lytic and lysogenic cycles
• Releases ~100 phages

Insertion Vector/Replacement Vector

• Used for DNA fragment cloning
• Includes different constructs based on different goals

In Vitro Packaging

• Proteins (gene E) create preheads
• Single-stranded DNA inserted
• Proteins assemble to form a complete phage

Agrobacterium tumefaciens-based Vectors

• Sizes over 200kb
• Tumour inducing (Ti) Plasmids transferred to Ti plasmid
• 25 kb - 239 kb
• Right and left borders
• Processing and Transfer of T-DNA occurs through Vir genes
  • Capable of autonomous replication
• Tumour formation by T-DNA integration
• PTI-based vectors used for reduction in size
• Removing opines
• Normal cant replicate in yeast cells
• Insertion/deletion of selectable markers
• Modified size of vector can be achieved

Cosmids

• Hybrid vector
• Lambda phage vector
• Plasmid vector
• 30-52kb cloning limit

Expression Vectors

• Strong promoters (e.g., IPTG)
• Translation initiation sequences
• Elements for expression (e.g., Kozak consensus sequence)
• Purification tags (e.g., His tag)
• Fusion proteins
• Antibiotic selection marker

Methods/Tools

• Restriction enzymes
• Ligases
• Polymerases (Taq polymerase)
• PCR
• Southern Blotting
• Northern Blotting
• Western Blotting
• Gene Cloning, etc.

This quiz covers the essentials of artificial chromosome vectors, including DNA assembly in vitro, their characteristics, and types such as BACs and YACs. Learn about the components and applications of these vectors in cloning and genome sequencing.