12 an intoduction to genetic analysis.docVIP

Chapter 12 Recombinant DNA TechnologyKey Concepts Recombinant DNA is made by splicing a foreign DNA fragment into a small replicating molecule (such as a bacterial plasmid), which will then amplify that fragment along with itself and result in a molecular clone of the inserted DNA. Restriction enzymes cut DNA at specific target sites, resulting in defined fragments with sticky ends suitable for insertion into a vector that has been cut open by the same enzyme. A collection of DNA clones that encompasses the entire genome of an organism is called a genomic library. An individual DNA clone can be selected from a library by using a specific probe for the DNA or its protein product or by its ability to transform a null mutant. DNA fragments of different sizes produced by restriction-enzyme digestion can be fractionated because they migrate to different positions on an electrophoretic gel. RNA or restriction-enzyme-cut DNA molecules that have been fractionated by size in an electrophoretic gel can be probed to detect a specific molecule. Restriction-enzyme target sites can be mapped, providing useful markers for DNA manipulation. A gene can be found by testing overlapping clones radiating outward from a linked marker. After a gene has been cloned, its nucleotide sequence can be determined, and the sequence can be used to study gene function and evolution. A pair of replication primers spanning a DNA sequence can be used to amplify that sequence for isolation. Introduction The goal of genetics is to study the structure and function of genes and genomes. Since Mendels time, genes have been identified by observing standard phenotypic ratios in controlled crosses. Clues about gene function first came from correlating specific mutations with enzyme and other protein deficiencies. The correlation of mutant sites within a gene with amino acid substitutions in the appropriate protein led to a better understanding of gene structure and function. To these ideas were added discov