Map-based or Positional Cloning of Genes
If you have identified markers that is flank your gene and identified a clone to which the markers reside, you are on your way to determining where that gene resides. What you are going to do is a procedure termed chromosomal walking. This involves identifying the clone to which your markers reside and mapping the STS sites on that clone in your populaton. If the linkage distance does not improve, you next go to STS sites located on adjacent clones. Your repeat this process until you have a markers associated with a clone that co-segregate with your gene. What this means is that whenever one allele of your geneis expressed, the markers associated with that allele is also present, that isthe population does not show recombination. To speed the cloning process, it is best to have a marker that is tightly linked to the gene with which your are working. Therefore you will not have to do alot of additional screening.
Because you have your gene flanked on a single clone between two markers, you now know that the gene must be between those two markers. If the clone has been sequenced, you look for possible genes in that region. A gene would be recognized by looking for open reading frames (ORFs), sequences that most likely will encode a gene product. In the best situation, only a single ORF is found, but this often is not the case. Usually several possible ORFs are found.
What is next done depends upon your species. For plants, a direct proof is possible. The candidate gene is introduced into a genotype that is mutant for the gene by a genetic engineering technique called plant transformation. The transgenic plant (the product of plant transformation) is analyzed, and if the gene that is introduced confers the wild type phenotype then you know you have cloned the gene.
For species such as humans, the development of transgenics is not possible. Therefore a mutational analysis is required. A library is made of individuals expressing the mutated gene. The ORFs are used as probes to select complementary sequences from the library containing mutant DNA. Next the ORFs are sequenced and compared with the ORFs from the normal DNA library. That ORF from the mutant library which is a different from its counterpart in the normal library is a strong candidate for the gene. Expression studies must be performed to further confirm the conclusion. That is, the clone should hybridize to mRNAs in tissues in which the gene is expressed, and it should show some different pattern of expression. Again, this is the simple scenariio. As you can imagine, much more complex analyses may be necessary to confirm that a gene has been cloned.
Copyright © 1997. Phillip McClean