The gene for color blindness in humans is on the X chromosome. The biochemical d

Question

The gene for color blindness in humans is on the X chromosome. The biochemical defect is unknown. Can you devise a method by which the gene might be cloned? What kind of library would you use, and how would you screen it? so i was thinking i could use a genomic library, and screening it by chromosome walking, but how would i obtain the clone? i think i could be able to use the genes on the chromosome as the probe as it is similar to the gene i am looking for, but im confused only on how i can obtain a clone to search for the color blindness gene? thank you!

Explanation / Answer

Gene cloning is the act of making copies, or clones, of a single gene. Once a gene is identified, clones can be used in many areas of biomedical and industrial research. Genetic engineering is the process of cloning genes into new organisms, or altering the DNA sequence to change the protein product. Genetic engineering depends on our ability to perform the following essential procedures. 1. Polymerase Chain Reaction The discovery of thermostable DNA polymerases, such as Taq Polymerase, made it possible to manipulate DNA replication in the laboratory and was essential to the development of PCR. Primers specific to a particular region of DNA, on either side of the gene of interest, are used, and replication is stopped and started repetitively, generating millions of copies of that gene. These copies can then be separated and purified using gel electrophoresis. 2. Restriction Enzymes The discovery of enzymes known as restriction endonucleases has been essential to protein engineering. These enzymes cut DNA at specific locations based on the nucleotide sequence. Hundreds of different restriction enzymes, capable of cutting DNA at a distinct site, have been isolated from many different strains of bacteria. DNA cut with a restriction enzyme produces many smaller fragments, of varying sizes. These can be separated using gel electrophoresis or chromatography. 3. Electrophoresis Purifying DNA from a cell culture, or cutting it using restriction enzymes wouldn't be of much use if we couldn't visualize the DNA - that is, find a way to view whether or not your extract contains anything, or what size fragments you've cut it into. One way to do this is by gel electrophoresis. Gels are used for a variety of purposes, from viewing cut DNA to detecting DNA inserts and knockouts. 4. Join Two Pieces of DNA In genetic research it is often necessary to link two or more individual strands of DNA, to create a recombinant strand, or close a circular strand that has been cut with restriction enzymes. Enzymes called DNA ligases can create covalent bonds between nucleotide chains. The enzymes DNA polymerase I and polynucleotide kinase are also important in this process, for filling in gaps, or phosphorylating the 5' ends, respectively. 5. Selection of Small Self-Replicating DNA Small circular pieces of DNA that are not part of a bacterial genome, but are capable of self-replication, are known as plasmids. Plasmids are often used as vectors to transport genes between microorganisms. In biotechnology, once the gene of interest has been amplified and both the gene and plasmid are cut by restriction enzymes, they are ligated together generating what is known as a recombinant DNA. Viral (bacteriophage) DNA can also be used as a vector, as can cosmids, recombinant plasmids containing bacteriophage genes. 6. Method to Move a Vector into a Host Cell The process of transferring genetic material on a vector such as a plasmid, into new host cells, is called transformation. This technique requires that the host cells are exposed to an environmental change which makes them "competent" or temporarily permeable to the vector. Electroporation is one such technique. The larger the plasmid, the lower the efficiency with which it is taken up by cells. Larger DNA segments are more easily cloned using bacteriophage, retrovirus or other viral vectors or cosmids in a method called transduction. Phage or viral vectors are often used in regenerative medicine but may cause insertion of DNA in parts of our chromosomes where we don't want it, causing complications and even cancer. 7. Methods to Select Transgenic Organisms Not all cells will take up DNA during transformation. It is essential that there be a method of detecting the ones that do. Generally, plasmids carry genes for antibiotic resistance and transgenic cells can be selected based on expression of those genes and their ability to grow on media containing that antibiotic. Alternative methods of selection depend on the presence of other reporter proteins such as the x-gal/ lacZ system, or green fluorescence protein, which allow selection based on color and fluorescence, respectively. http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/micro10.swf::Steps%20in%20Cloning%20a%20Gene

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