BIOLOGY Volume 3 - A Textbook of General Biology - 2004

25. APPLIED GENETICS

25.1. Bacterial Genetic Engineering

25.1.3. Step 2. Insertion of Genes into a Vector

As explained above, plasmid DNA or phage DNA is most commonly used as a vector. Let us first examine the Procedure for insertion into plasmid DNA. Essentially, the same protocol applies to phage DNA.

Plasmid DNA

Circular plasmid DNA molecules in Bacteria are much smaller than the main chromosomal DNA, making them easy to separate. Bacterial Cells are lysed and centrifuged. This causes the chromosomal DNA to pellet, while the plasmid DNA remains in the supernatant. The plasmid DNA is purified prior to restriction enzyme Digestion (Fig. 25.1).

If a restriction enzyme was used to isolate the donor DNA (e.g., via the "shotgun" method), the plasmid DNA must be digested with the exact same enzyme. The restriction fragments of the donor DNA, including the fragment containing the target Gene, are then mixed with the plasmid DNA. Upon mixing, they join via complementary sticky ends. For example, a sticky end with the sequence —AATT will pair with a complementary —TTAA sticky end. Initially, the connection is held together by Hydrogen Bonds, but after The addition of an enzyme called DNA ligase, stable phosphodiester bonds are formed.

If cDNA or a synthetic gene is used as the donor DNA (refer to the first two gene Isolation Methods described above), the DNA must be inserted into the vector following the protocol shown in Fig. 25.6. The same procedure is required if an enzyme that produces blunt ends was used in the shotgun method.

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Fig. 25.6. Addition of sticky ends to a DNA fragment with blunt ends prior to insertion into a vector to create a recombinant DNA molecule.

Phage Vector

The advantage of using phages as vectors is The ability to clone much larger DNA fragments than Plasmids can carry. The λ phage is most frequently used for this purpose (Fig. 2.20). A non-essential portion of the phage DNA is replaced with the DNA to be cloned. Because this segment is not required for phage METABOLISM/36.html">DNA Replication within the host bacterial Cell, cloning is not disrupted. This procedure is outlined schematically in Fig. 25.7.

Donor DNA obtained via the "shotgun" method is frequently used in this process.

Fig. 25.7. Incorporation of DNA into a phage vector.