Define the characters of DNA vectors. Explain with diagrams how these vectors function in a variety of situations.

Characters of DNA Vectors

DNA vectors possess several key characteristics that make them suitable for gene cloning and delivery:

• Origin of Replication (ori): Essential for the vector to replicate independently within the host cell.
• Selectable Marker: A gene conferring resistance to an antibiotic or providing a nutritional advantage, allowing for the identification of host cells that have taken up the vector.
• Multiple Cloning Site (MCS): A short DNA sequence containing recognition sites for several restriction enzymes, facilitating the insertion of foreign DNA.
• Small Size: Generally, smaller vectors are easier to manipulate and introduce into host cells.
• Copy Number: The number of copies of the vector present in each host cell. High copy number vectors yield more of the cloned gene.

Types of DNA Vectors and their Functioning

1. Plasmids

Plasmids are extrachromosomal, circular DNA molecules found in bacteria and some eukaryotes. They are widely used for cloning small DNA fragments (up to 10 kb).

Functioning: A gene of interest is inserted into the MCS of the plasmid using restriction enzymes and DNA ligase. The recombinant plasmid is then introduced into bacterial cells (transformation). Cells containing the plasmid are selected using antibiotic resistance. The plasmid replicates within the bacteria, producing multiple copies of the inserted gene.

2. Bacteriophages (Phage Vectors)

Bacteriophages are viruses that infect bacteria. They can accommodate larger DNA fragments (up to 20 kb) than plasmids.

Functioning: The phage DNA is modified to remove non-essential genes, creating space for the foreign DNA. The recombinant phage infects bacterial cells, injecting the DNA. The phage replicates within the bacteria, producing more phage particles carrying the cloned gene.

3. Cosmids

Cosmids are hybrid vectors combining features of plasmids and bacteriophages. They can carry even larger DNA fragments (up to 45 kb).

Functioning: Cosmids utilize the cos sites from bacteriophage lambda to package DNA into phage particles. These particles then infect bacteria, where the cosmid DNA circularizes and replicates as a plasmid.

4. Phagemids

Phagemids are plasmids that contain a phage origin of replication. They can be propagated as plasmids in bacteria and packaged into phage particles.

Functioning: Similar to cosmids, phagemids can be packaged into phage particles for efficient delivery into bacterial cells. They offer advantages in terms of ease of manipulation compared to cosmids.

5. Artificial Chromosomes (BACs, YACs, MACs)

These vectors are designed to clone very large DNA fragments (over 100 kb).

• Bacterial Artificial Chromosomes (BACs): Based on the F plasmid of E. coli, capable of carrying fragments up to 300 kb.
• Yeast Artificial Chromosomes (YACs): Based on yeast chromosomes, can carry fragments up to 1 Mb.
• Mammalian Artificial Chromosomes (MACs): Based on mammalian chromosomes, can carry very large fragments and maintain complex genomic features.

Functioning: These vectors are introduced into host cells (bacteria for BACs, yeast for YACs, mammalian cells for MACs) where they replicate and maintain the large DNA fragments.

Vector Type	Insert Size (kb)	Host Cell	Advantages	Disadvantages
Plasmid	Up to 10	Bacteria	Easy to manipulate, high copy number	Small insert size
Bacteriophage	Up to 20	Bacteria	Larger insert size than plasmids	More complex manipulation
Cosmid	Up to 45	Bacteria	Larger insert size, efficient packaging	Can be unstable
BAC	Up to 300	Bacteria	Very large insert size, stable	Lower transformation efficiency
YAC	Up to 1000	Yeast	Extremely large insert size	Chimeric rearrangements, instability