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BIOL 3110
Peter Cheung

Oct 22, 2009 Restriction Fragment Length Polymorphisms (RFLP’s) -This method of analysis does not rely on the mutation having a phenotype change, some genes can be polymorphic, therefore, this is a much more useful and accurate method. (refer to fig 4.1 Genes IX) -Restriction site polymorphisms change the sequence of the code and can possibly create or destroy restriction site. -The higher the recombination frequency the greater the distance the allele is from the center of the gene and vice versa. ***refer to previous notes for clarification*** -Gene targeting is a useful tool for identifying the probability of phenotype appearing (genetic diseases); it can also be used for isolating genes of interest. Determining Gene Structure -The first step is to identify the size of the DNA insert. -Once the size of the insert is determined the genome of interest needs to be mapped via restriction enzymes. -The mapped genome can now provide the location of the gene and the actual gene can then be analyzed by Southern Blot Analysis. -The next step is to perform Heteroduplex mapping: 1)the isolated fragment is mixed with either sscDNA or mRNA 2)anneal so that a hybrid molecule is created 3)the hybrid is then placed on a grid and searched for heteroduplexes, loops on the map will represent introns. *if the hybrid does not anneal properly then it means only fragment of the gene was isolated. Interrupted Gene -These are uncommon occurrences but can be found everywhere -bacteria and phages -yeast more often than not contain introns -higher eukaryotes have introns in most genes. -All classes of genes may have introns (mRNA, rRNA, tRNA), mitochondrial and chloroplast genes also contain introns. -Related genes may have similar exon/intron arrangements, α & β globin genes are usually familiar too. -Pseudo-genes are non-functional genes that have the same arrangement as a wild type gene but it is slowly decaying due to disuse. -Exon sequences are conserved but intron sequences vary over evolution. The isolation of exons allows us to isolate and identify genes. -Exon coding for proteins: -ORF: open reading frames, doesn’t contain stock codons for proteins -should be found in related animals Chromosome Walking -Refer to previous notes -As the chromosome walk is performed the gene needs to be tested for exons, there are a number of ways to do so: 1)Zoo Blot – essentially a Southern Blot of genomic DNA of closely related animals. The related genomes are then digested with restriction
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