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DNA TECHNOLOGY DNA Cloning Gene Cloning: Producing multiple identical copies of a gene. Recombinant DNA: Genes or DNA from two difference sources are combined into one molecule. Genetic Engineering: Methods to create recombinant DNA. Plasmids as Vectors Plasmids: self replicating circular units of DNA that can be used to carry cloned genes. Cloning Vector: DNA molecule that can carry foreign DNA. • Plasmids are a type of cloning vector. Restriction enzymes are used to cut open the plasmid. The same restriction enzyme is used to cut the gene out of its original source. Complimentary sticky ends allows the sticky ends to hydrogen bond together. Ligase is used to reseal the phosphodiester bonds of the DNA backbone. Transforming Bacteria with the Cloning Vector Transformation: the uptake of DNA from the environment. • Plasmids containing the gene of interest can be introduced into bacteria which then multiply and produce clones that also carry the gene. • These clones a can produce the gene product in large quantities. Inserting the Vector Plasmids that carry antibiotic resistant genes are used. • Bacteria that acquire the vector can grow on growth medium containing antibiotics where other bacteria cannot. • This allows selection for bacteria only carrying the vector. The gene of interest is inserted in the middle of another gene and disrupts its function. Screening for the Bacteria Containing the Cloned Gene In this particular example the bacteria used are lacZ-. • This means they are unable to metabolize lactose because they have a mutated lacZ gene. • When they acquire the vector they can now break down lactose. • X-gal is a supplement that creates a blue colored product when lactose is broken down. • The gene of interest is inserted into the lacZ gene and renders it inactive. When the insert is placed in the vector some of the vectors take up the insert and others do not. Screening for the Bacteria Containing the Cloned Gene How do you distinguish between the bacteria that have the insert and ones that don’t? • The bacteria are plated out on a medium containing X-gal. • Bacteria that have an intact lacZ gene will hydrolyze X-gal and produce a blue product. • Bacteria that contain the insert have a disrupted lacZ gene and cannot break down Xgal so the colonies remain white. The Polymerase Chain Reaction A process that produces many identical copies of the same gene. • Allows multiple copies to be produced when there is very little DNA available. DNA from fossils DNA from a crime scene • The Process involves a DNA primer. Remember DNA polymerase can only nucleotides to a preexisting DNA molecule. The primer is homologous to the gene of interest and extends the DNA complimentary to the gene. Polymerase Chain Reaction DNA Analysis What if we wanted to know the sequence of gene? • To compare allele differences associated with heredity disorders. • To compare closely related two species are. • To determine whether or not some one committed a crime. Restriction Fragment Analysis Uses electrophoresis to separate DNA fragments after they have been digested with restriction enzymes. • Mutated forms of an allele are going to exhibit a different band pattern on the gel. This is because the restriction sites are in different places on the allele yielding fragments of DNA that vary from the bands of the non mutated allele. Scientists can match band patterns on the gel but only know the relative sizes of the DNA fragments. They do not know the actual DNA sequence. Electrophoresis The Gel The Southern Blot This procedure allows the identification of specific sequences of DNA. • An imprint of the DNA is made on a filter that is placed on top of the gel. • The DNA on the filter is now “denatured” and made single stranded. • The filter is then incubated with a labeled probe (radioactive) that is complimentary to the gene of interest. Since the DNA on the filter is single stranded it will bind to the complimentary sequences of the probe. • The filter is washed to remove excess probe that is not bound and the filter is then exposed to film. • The film shows where the radioactive probe was complimanty to the gene. This is known as a autoradiograph. The Southern Blot Restriction Fragment Length Polymorphisms RFLP Markers: Adjacent to genes are satellite sequences that do not code for anything but vary between individuals. • Because the restriction sites are in different places within the RFLP sequences each person has a DNA “fingerprint”. Even though the sequence of the gene itself is the same from person to the next the RFLP sequences account for different banding patterns. DNA can indicate the presence of an individual at the scene of a crime The Autoradiograph • http://www.dnalc.org/ddnalc/resources/pcr.h tml