7.3.1.1 The procedure of Recombinant DNA

7.3.1.1 The procedure of Recombinant DNA

Step1.Cell membrane dissolution: E.coli cells of a specific strain are placed in a solution that dissolves cell membranes, thus releasing the contents of the cells.

Step2. Isolation of plasmid fraction: The released cell components are separated into fractions, one fraction being the plasmids. The isolated plasmid fraction is the material used in further steps.

Step3. Cleavage of plasmid DNA: A special enzyme, called a restriction enzyme, is used to cleave the double-stranded DNA of a circular plasmid. The result is a linear (noncircular) DNA molecule.

Step4. Gene removal from another organism: The same restriction enzyme is then used to remove a desired gene from a chromosome of another organism.

Step5. Gene-plasmid splicing: The gene (from Step 3) and the opened plasmid (from Step3) are mixed in the presence of the enzyme DNA ligase, which spliced the two together. This splicing, which attaches one end of the gene to one end of the opened plasmid and attaches the other end of the gene to one end of the plasmid, results in an altered circular plasmid(the recombinant DNA).

Step6. Uptake a recombinant DNA: The altered plasmids (recombinant DNA) are placed in a live E.coli culture, where they are taken up by the E.coli bacteria. The E.coli culture into which the plasmids are placed need not be identical to that from which the plasmids were originally obtained.

It can be noted from step 3 that the conversion of a circular plasmid into a linear DNA molecule requires a restriction enzyme. A restriction enzyme is an enzyme that recognizes specific base sequences in DNA and cleaves the DNA in a predictable manner at the sequences. The discovery of restriction enzymes made genetic engineering possible.

Restriction enzymes occur naturally in numerous types of bacterial cells. Their functions are to protect the bacteria from invasion by foreign DNA by catalyzing the cleavage of the invading DNA. The term restriction relates to such enzymes placing a “Restriction” on the type of DNA allowed into the bacterial cells.

The restriction enzyme works in the following way. Consider one restriction enzyme that cleaves DNA between G and A bases in the 5’-to-3’ direction in the sequence G-A-A-T-T-C. This enzyme will cleave the double-helix structure of a DNA molecule in the manner shown in Figure .

During this process the double helix is not cut straight across but the individual strands are cut at different points, giving a staircase cut. (Both cuts must be between G and A in the 5’-to-3’ direction). This staircase cut leaves unpaired bases on each cut strand. These ends with unpaired bases are called “sticky ends” because they are ready to “sticky to”(pair up with) a complementary section of DNA if they can find one.

If the same restriction enzyme used to cut a plasmid is also used to cut a gene from another DNA molecule, the sticky ends of the gene will be complementary to those of the plasmid. This enables the plasmid and gene to combine readily, forming a new, modified plasmid molecule.

This modified plasmid molecule is called recombinant DNA. In addition to the newly spliced gene, the recombinant DNA plasmid contains all of the genes and characteristics of the original plasmid. Step 6 involves inserting the recombinant DNA(modified plasmids)back into E.coli cells. The process is called transformation. Transformation is the process of incorporating recombinant DNA into a host cell.

The transformed cells then reproduce, resulting in large number of identical cells called clones. Clones are cells with identical DNA that have descended from a single cell. Within a few hours, a single genetically altered bacterial cell can give rise to thousands of clones. Each clone has the capacity to synthesize the protein directed by the foreign gene it carries.

Researches are not limited to selection of naturally occurring genes for transforming bacteria. Chemists have developed nonenzymatic methods of linking nucleotides together such that they can construct artificial genes of any sequence they desire. Instruments are now available that can be programmed by a microprocessor to synthesize any DNA base sequence automatically. The operator merely enters a sequence of desired bases, starts the instrument, and returns later to obtain the product. Such flexibility in manufacturing DNA has opened many doors, accelerated the pace of recombinant DNA research, and redefined the term designer genes.

Last modified: Wednesday, 22 February 2012, 9:30 AM