4.3 Vectors and gene cloning strategies

Vectors are essential tools for genetic engineering, allowing scientists to insert and manipulate DNA in host organisms. From plasmids to YACs, these molecular vehicles come in various sizes and shapes, each with unique advantages for different cloning applications.

Cloning strategies like cDNA and genomic DNA cloning help researchers study gene expression and structure. These techniques, combined with specialized vectors, enable the creation of libraries and the production of recombinant proteins, advancing our understanding of genetics and biotechnology.

Cloning Vectors

Plasmids and Bacteriophages

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• Plasmids are small, circular, double-stranded DNA molecules found in bacteria that replicate independently of the host genome
  • Commonly used plasmids include pBR322 and pUC19
• Bacteriophages are viruses that infect bacteria and can be used as cloning vectors
  • Lambda phage and M13 phage are popular choices for cloning
• Both plasmids and bacteriophages have been engineered to contain multiple cloning sites (MCS) which facilitate the insertion of foreign DNA fragments

Cosmids and Bacterial Artificial Chromosomes (BACs)

• Cosmids are hybrid vectors that combine features of plasmids and bacteriophages
  • Can accommodate larger DNA inserts (up to 45 kb) compared to plasmids
  • Contain cos sites derived from lambda phage for efficient packaging and delivery into host cells
• Bacterial artificial chromosomes (BACs) are large, low-copy number vectors based on the F-factor plasmid of E. coli
  • Can carry inserts up to 300 kb in size
  • Maintain stable propagation of large DNA fragments with minimal risk of rearrangement

Yeast Artificial Chromosomes (YACs)

• Yeast artificial chromosomes (YACs) are vectors designed for cloning in yeast cells (Saccharomyces cerevisiae)
  • Can accommodate extremely large DNA inserts (up to 2 Mb)
  • Contain essential elements for replication and segregation in yeast, such as telomeres, centromeres, and autonomous replication sequences (ARS)
• YACs are useful for constructing genomic libraries and studying large, complex genomes
  • However, they are prone to chimerism and instability, which can complicate their use

Specialized Vectors

Expression Vectors

• Expression vectors are designed to enable the production of recombinant proteins in host cells
  • Contain promoter and terminator sequences to drive transcription of the inserted gene
  • May include tags (His-tag, GST-tag) for easy purification of the expressed protein
• Different expression systems are available for various host organisms (E. coli, yeast, mammalian cells, insect cells)
  • Choice depends on factors such as post-translational modifications, protein solubility, and yield

Shuttle Vectors

• Shuttle vectors can replicate in two or more different host species, allowing for transfer of genetic material between them
  • Commonly used to move DNA between E. coli and other organisms (yeast, mammalian cells, plants)
• Contain multiple origins of replication and selectable markers functional in each host
  • For example, a yeast-E. coli shuttle vector may have an ori for E. coli, an ARS for yeast, and antibiotic resistance genes for selection in both hosts
• Facilitate the manipulation of DNA in E. coli before introduction into the final host organism for expression or functional studies

Cloning Strategies

cDNA Cloning

• cDNA (complementary DNA) cloning involves the synthesis of DNA from mature mRNA templates using reverse transcriptase
  • Captures the expressed portion of the genome without introns
  • Useful for studying gene expression and creating expression libraries
• Steps in cDNA cloning:
  1. Isolation of total RNA or mRNA from cells or tissues
  2. First-strand cDNA synthesis using reverse transcriptase and an oligo(dT) primer
  3. Second-strand cDNA synthesis using DNA polymerase I and RNase H
  4. Insertion of the double-stranded cDNA into a cloning vector
• cDNA libraries represent the genes expressed in a particular cell type or developmental stage

Genomic DNA Cloning

• Genomic DNA cloning involves the insertion of fragments from an organism's entire genome into a cloning vector
  • Captures both coding and non-coding regions, including introns and regulatory sequences
  • Useful for studying gene structure, regulation, and evolution
• Steps in genomic DNA cloning:
  1. Isolation of high-molecular-weight genomic DNA from cells or tissues
  2. Fragmentation of the DNA using restriction enzymes or mechanical shearing
  3. Size-selection of DNA fragments (if desired) by gel electrophoresis
  4. Ligation of the DNA fragments into a suitable cloning vector
• Genomic libraries provide a comprehensive representation of an organism's entire genome
  • Screened using probes or PCR to identify clones containing specific genes or regions of interest