Gene Splicing
The term gene splicing refers to molecular biochemical techniques used to attach different DNA molecules to one another. The resulting recombinant DNA can be used for many subsequent purposes. Gene splicing, more commonly referred to as cloning, is a basic technique widely used in genetic, biochemical, and increasingly, physiological studies.
A simple and common splicing method relies on the use of restriction endonucleases to cleave the DNA molecules. The fragment of DNA of interest must then be entered into a vector molecule in order to make many copies of the DNA. The vector, generally a bacterial plasmid, is also cleaved with an enzyme that will leave ends that can be annealed to the fragment ends, usually the same enzyme or combination of enzymes used to cleave the fragment.
Many restriction enzymes leave overhanging single-stranded ends that are complementary to each other on the different DNA molecules. These are known as sticky ends, because they readily reanneal under the appropriate conditions. Other enzymes leave blunt-ended fragments that can be ligated to other DNA molecules with blunt ends. The use of two enzymes that leave different sticky ends is convenient for cloning the DNA in a directional manner, so that one end of the DNA is more likely to face in the desired direction. If overhangs are made with the same enzyme, or if the ends are blunt, then the fragment may insert in either direction with respect to the vector. The orientation of the inserted fragment must then be confirmed, either by sequencing or by digesting the resulting plasmid with enzymes that will cut at known sites in the DNA.
After the initial digestions, the two DNA molecules, fragment and vector, are covalently attached by virtue of their complementary overhangs under annealing conditions by the action of another enzyme, DNA ligase. The ligation mixture is then transformed into bacterial cells, normally a strain of Escherichia coli. The plasmid usually contains a drug resistance gene. Therefore, only bacteria harboring the new plasmid will survive when grown in media containing the appropriate antibiotic drug. Ampicillin, kanamycin, and chloramphenicol are drugs commonly used to select bacterial colonies with the recombinant DNA.
Because the plasmid also contains a bacterial origin of replication, it is replicated many times in the bacterial cells. Microgram quantities of the plasmid can then be purified from as little as a milliliter of culture of bacteria harboring the recombinant plasmid. The large quantities of recombinant DNA made can then be used for many subsequent purposes.
The fragment is often cloned such that it can be dropped out of the plasmid by cleavage with the same enzyme used in the original cloning steps. It can then be used for multiple purposes, for example, as a probe in Southern and northern blot analyses. An interesting use of gene splicing involves placing one gene under the control of a promoter different from its own. Thus, for example, high levels of the RNA may be expressed when controlled by a highly active promoter, or the RNA may be preferentially expressed in certain cells or tissues or only under specific biotic or abiotic conditions. Alternatively, a specific promoter may be used to drive expression of a reporter transcript to examine where and under what conditions the promoter is active. Popular reporters include beta-galacturonidase, green fluorescent protein, and luciferase, all of which allow visual localization of the protein made from the recombinant transcript. A gene of interest may also be expressed to high levels in a heterologous system; for example, a mouse gene may be expressed in bacterial cells in order to purify large quantities of the protein encoded by the mouse gene for functional, biochemical, or structural studies.
In certain uses, gene splicing may also refer to the removal of introns from pre-mRNA transcripts, leaving only exon sequences in the mature mRNA molecule.
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