RNA Dependent-DNA Polymerase and cDNA Synthesis

RNA Dependent-DNA Polymerase (Reverse Transcriptase)

• Reverse transcriptase (RT) synthesizes single-stranded DNA from an mRNA template.
• Like regular DNA polymerases, RT needs a primer to start.
• RT possesses 5'→3' exonuclease activity and 3'→5' exonuclease activity, specifically degrading RNA in DNA-RNA hybrid molecules.
• RT extracted from RNA tumor viruses is often used to convert mRNA to dsDNA, for insertion into prokaryotic vectors.

cDNA Synthesis

• First, cDNA is synthesized.
• Next, the RNA is degraded by alkali or ribonuclease H.
• A second strand of cDNA is synthesized using the Klenow fragment of DNA polymerase I or RT itself.
• The cDNA strand acts as a primer and template, forming a hairpin structure.
• Crucially, RT has no proofreading ability.

Cloning and Expression Vectors

Plasmid Vectors

• Plasmids are named using a system of uppercase letters and numbers (e.g. pBR322). The "p" denotes plasmid.
• Some early plasmids had low copy numbers (one or two copies per cell), while others, like pUC18 and pUC19, have high copy numbers (>500 copies per cell).
• High copy number plasmids replicate independently of the host chromosome (relaxed control), whereas stringent control plasmids' replication is tied to the host chromosome.
• An ideal plasmid vector has an origin of replication (ori), selection marker(s) for identifying successful host cells (e.g antibiotic resistance), a multiple cloning site (MCS) for inserting foreign DNA, and efficient extraction from the host cell.
• Techniques like single or twin antibiotic resistance and blue-white screening aid in selecting successful vectors.

Other Vector Types

• Bacteriophage vectors, like lambda (λ), can replicate within bacterial cells, facilitating the insertion of more extensive DNA fragments (>10kbp).
• Cosmids combine the benefits of plasmid (easy propagation) and phage (high capacity) vectors. Cosmids have a cos site, enabling packaging into phage heads, suitable for carrying larger DNA fragments (33-47 kbp).
• Bacterial artificial chromosomes (BACs) are based on the fertility plasmid (F plasmid) allowing the insertion of large DNA fragments (>50 kbp).
• Yeast artificial chromosomes (YACs) can accommodate exceptionally large inserts (100-1000 kbp).

Expression Vectors

• Expression vectors are designed for expressing a target gene at high levels. Typically, they contain a promoter to initiate transcription.
• They use efficient T7 RNA polymerase systems or other protein expression strategies.
• Cloning a target gene into the vector allows its expression in bacterial hosts, which can yield large amounts of the encoded protein.