Recombinant DNA Technology
Recombinant DNA technology involves combining DNA from different sources to create new combinations of genetic material. This technology underpins modern genetic engineering and biotechnology, enabling the production of medically important proteins, genetically modified organisms, and gene therapy. This lesson covers restriction enzymes, ligation, vectors, transformation, and screening.
What Is Recombinant DNA?
Key Definition: Recombinant DNA is DNA that has been artificially created by combining DNA from two or more different sources (often from different species).
The basic process involves:
- Isolating the gene of interest from one organism.
- Cutting the gene and a vector using restriction enzymes.
- Joining the gene into the vector using DNA ligase.
- Introducing the recombinant vector into a host cell (transformation).
- Selecting host cells that have taken up the recombinant DNA.
Restriction Enzymes (Restriction Endonucleases)
Key Definition: Restriction enzymes are enzymes that cut DNA at specific base sequences called recognition sites (or restriction sites). They were originally discovered in bacteria where they function as a defence mechanism against bacteriophage (viral) DNA.
Key Features
- Each restriction enzyme recognises a specific palindromic sequence (typically 4–8 base pairs long). A palindromic sequence reads the same on both strands in the 5' to 3' direction.
- Example: The restriction enzyme EcoRI recognises the sequence:
- 5'-G|AATTC-3'
- 3'-CTTAA|G-5'
- (The vertical lines indicate where the enzyme cuts.)
Types of Cut
- Sticky ends — the enzyme cuts the two strands at different positions within the recognition site, leaving short single-stranded overhangs (usually 2–4 bases). These overhangs can form hydrogen bonds with complementary sticky ends produced by the same restriction enzyme, facilitating the joining of DNA fragments.
- Example: EcoRI produces sticky ends with the overhang 5'-AATT-3'.
- Blunt ends — the enzyme cuts both strands at the same position, leaving no overhangs. Blunt ends can be joined but less efficiently because there are no complementary single-stranded regions to guide base pairing.
Exam Tip: Sticky ends are preferred in genetic engineering because they allow complementary base pairing between the gene insert and the vector, making ligation more efficient. Always specify that the same restriction enzyme must be used to cut both the gene and the vector to produce compatible sticky ends.
DNA Ligase
Key Definition: DNA ligase is an enzyme that catalyses the formation of phosphodiester bonds between the sugar-phosphate backbones of adjacent DNA fragments, permanently joining them together.
- After the gene of interest and the vector have been cut with the same restriction enzyme, they are mixed together.
- Complementary sticky ends base-pair by hydrogen bonding (this is temporary and weak).
- DNA ligase seals the gaps in the sugar-phosphate backbone, creating a continuous recombinant DNA molecule.
- Ligase can also join blunt ends, though this is less efficient and may require higher enzyme concentrations.
Vectors
Key Definition: A vector is a carrier molecule used to transfer a gene of interest into a host cell. Vectors must be capable of replicating within the host cell.
Plasmid Vectors
- Plasmids are small, circular, double-stranded DNA molecules found naturally in bacteria.
- They replicate independently of the bacterial chromosome.
- Key features of plasmid vectors:
- Origin of replication (ori) — allows the plasmid to replicate autonomously in the host cell.
- Antibiotic resistance genes — used as selectable markers to identify bacteria that have taken up the plasmid.
- Multiple cloning site (MCS) — a short region containing recognition sites for several different restriction enzymes, providing flexibility in cloning.
- Plasmids can typically carry inserts of up to ~10 kilobases (kb).
Bacteriophage Vectors
- Bacteriophages (phages) are viruses that infect bacteria.
- Part of the phage DNA (non-essential genes) can be replaced with the gene of interest.
- The recombinant phage infects bacteria and introduces the foreign DNA.
- Phage vectors can carry larger DNA inserts (up to ~25 kb for lambda phage).
- Infection is often more efficient than transformation with plasmids.
Other Vectors
- Yeast artificial chromosomes (YACs) — can carry very large inserts (up to 1,000 kb); used in genome mapping projects.
- Cosmids — hybrid vectors combining features of plasmids and phage; can carry ~45 kb inserts.
- Viral vectors — modified viruses (e.g., adenoviruses, retroviruses, lentiviruses) used to deliver genes into mammalian cells, particularly in gene therapy.
Obtaining the Gene of Interest