Q.Explain the three steps carried out in the formation of recombinant DNA using the enzyme EcoRI.
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Start your 14-day free trial to unlock the full solution →The formation of recombinant DNA using EcoRI involves three precise steps: cutting both the source DNA and the vector DNA with the same restriction enzyme, allowing the sticky ends to pair up, and then sealing the joined fragments with DNA ligase.
To understand how EcoRI helps build recombinant DNA, you first need to picture what this enzyme actually does. EcoRI is a restriction enzyme — a molecular scissors — that cuts DNA at a very specific recognition sequence: GAATTC. What makes it special is that it cuts the two strands of DNA in a staggered fashion, not straight across. This leaves short, single-stranded overhangs on each end, known as "sticky ends." These sticky ends are complementary to each other, meaning they can base-pair with any other DNA fragment that has been cut by the same enzyme.
Now, the goal of recombinant DNA technology is to take a piece of DNA from one organism (say, a human gene for insulin) and insert it into a small, self-replicating circle of DNA called a plasmid (the vector). The plasmid acts as a carrier, allowing the foreign gene to be copied and expressed inside a bacterial cell. EcoRI is the tool that makes this insertion possible.
Step 1: Cutting both DNAs with EcoRI
The first step is to treat both the source DNA (containing the gene of interest) and the plasmid vector with the same restriction enzyme — EcoRI. Because EcoRI recognises the same six-base sequence wherever it appears, it will cut both molecules at every GAATTC site. The source DNA gets fragmented into many pieces, each with sticky ends. The plasmid, being a small circular molecule, gets cut open at its single EcoRI site, converting it from a circle into a linear molecule. Crucially, both the plasmid and the gene-containing fragment now have identical, complementary sticky ends: one strand ends with AATT, and the other with TTAA.
The plasmid is usually engineered to have only one recognition site for EcoRI. If it had multiple sites, the plasmid would be cut into several useless pieces. A single cut simply linearises the circle, leaving it ready to accept a new piece of DNA.
Step 2: Mixing and annealing the sticky ends
Once both DNAs have been cut, they are mixed together in a test tube. Because the sticky ends are complementary, they will spontaneously pair up through hydrogen bonding between the exposed bases. The AATT overhang on the plasmid will base-pair with the TTAA overhang on the foreign DNA fragment, and vice versa. This process is called annealing. At this stage, the foreign DNA is held in place next to the plasmid DNA only by weak hydrogen bonds — it is not yet a permanent, covalently sealed molecule. Think of it as two puzzle pieces that have been loosely fitted together but not glued.
The annealing step is reversible. If the mixture is heated, the hydrogen bonds break and the fragments separate. That is why this step is usually carried out at a lower temperature (around 4°C to 16°C) to favour stable pairing. Also, note that the plasmid ends can also re-join with each other (self-ligation), which is why scientists often treat the plasmid with alkaline phosphatase to remove phosphate groups and prevent this unwanted event.
Step 3: Sealing with DNA ligase …
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