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Cloning Genes

Positional Cloning

Positional cloning starts with the classical methods developed at the turn of the twentieth century by Thomas Hunt Morgan, Alfred Sturtevant, and their colleagues, of genetically mapping a particular phenotype to a region of a chromosome. A detailed discussion of genetic mapping is beyond the scope of this section, but, in general, it is based on conducting genetic crosses between individuals with two different mutant traits and analyzing how often the traits occur together in the progeny of subsequent generations.

Genetic mapping provides a general idea of where a gene is located on a particular chromosome, but it does not identify the precise DNA sequence that encodes the gene. The next step is to locate the gene on what is called the physical map of the chromosome. A physical map is a high-resolution map of all the DNA sequences that make up a chromosome. One type of physical map is a restriction map, which depicts the order of DNA fragments produced when a large DNA molecule is cut with restriction endonucleases (restriction enzymes).

Restriction maps have been made for the complete genomes of several model genetic organisms, such as the fruit fly (Drosophila melanogaster), and the roundworm, (Caenorhabditis elegans). For these organisms, individual large DNA fragments—on the order of forty to one hundred thousand base pairs from the whole genome—have been cloned in bacterial plasmid vectors to make a "library" of the genome. Each fragment is mapped to a known The location of a gene can be found by cutting the DNA with restriction enzymes, inserting the fragments into bacteria, and then reintroducing them individually into mutant flies. position, but the identify of the gene or genes it contains is originally unknown. To identify the genes, a cloned fragment is introduced into a mutant fly or roundworm.

To pinpoint the location of a particular gene, a researcher can introduce one or several of the plasmid clones from the physical map that are in the general vicinity of the region on the genetic map where the gene is thought to lie into a mutant that is defective in the gene of interest. If the introduced DNA corrects the mutant's defect, that DNA probably contains a normal copy of the defective gene. But these large clones usually contain several genes. By further "trimming" the DNA into smaller subfragments and testing the ability of each subfragment to rescue mutants, the researcher can eventually home in on the gene. As further confirmation that this gene is the cause of the mutant phenotype, the researcher can isolate the corresponding gene from the mutant and determine its DNA sequence to see if Restrictive enzymes (scissors) cut a gene out of its normal chromosomal position. Other enzymes insert it into a plasmid, which is then introduced into a bacterium. Only those bacteria that took up the plasmid survive on the growth medium. These bacteria can then be grown in bulk to produce many gene copies. it contains a mutation (a DNA sequence alteration) relative to the normal gene sequence.

Additional topics

Medicine EncyclopediaGenetics in Medicine - Part 1Cloning Genes - Purposes Of Gene Cloning, Cloning Techniques, Importance For Medicine And Industry, Genomic Versus Cdna Clones