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AP Biology: Chapter 14 DNA Technologies Recombinant DNA methods – Restriction enzymes • Enzymes from bacteria • Used to cut DNA molecules in specific places • Enable researchers to cut DNA into manageable segments – Vector molecule • Carrier of DNA fragment into cell • Plasmids • Engineered viruses – Transformation • Uptake of foreign DNA into cells Splicing Foreign DNA into a Vector • Foreign DNA and plasmid DNA cut with same restriction enzyme • Produces linear molecules with complementary singlestranded ends • Recombinant DNA created by mixing so sticky ends pair • DNA ligase forms covalent bonds, linking the two fragments Plasmid Vector Video • Genetic probes – Segments of single-stranded DNA that can hybridize to complementary base sequences in target gene – Southern blot technique • Genomic library – Collection of DNA fragments that represent all the DNA in the genome • Chromosome library – All the DNA fragments in that specific chromosome • cDNA library – Produced using reverse transcriptase – Makes DNA copies of modified mRNA • Amplifying DNA in vitro by PCR • • • • Small amount of double-stranded DNA DNA precursors Specific nucleic acid primers Taq DNA polymerase – DNA is denatured – Primers attach to primer-binding site on each DNA strand – Each strand acts as template for DNA synthesis • DNA sequencing – Based on chain termination method – Yields information about • Structure of gene • Probable amino acid sequences of its encoded proteins Comprehension Check The gel at the right was produced from four samples of single-stranded DNA fragments that were incubated with radioactively labeled primer, DNA polymerase, the four nucleotides, and a different one of the four dideoxy nucleotides. 1. What is the sequence of nucleotides shown in this gel? GACTGAAGCTGTT ________________ 2. What is the sequence of nucleotides in the original single-stranded DNA fragment? CTGACTTCGACAA _________________ • Applications of DNA technology – Gene therapy – Tissue engineering – DNA typing • Transgenic organisms – Have a gene from some other organism inserted into their genome – Gene targeting • Adds precision to transgenic technology • “Knocks out” or “knocks in” gene of interest at particular chromosomal locus, where it trades places with an existing gene. • By causing a specific gene to be inactive in the mouse, and observing any differences from normal behavior or condition, researchers can infer its probable function Mouse on right is normal; mouse on left is transgenic animal expressing rat growth hormone • Safety guidelines – Safety concerns • Introduction of transgenic organisms into the environment • Health effects on humans from consuming genetically modified crops – Safety measures • Special facilities designed to hold pathogenic organisms • Science of risk assessment: the benefits must outweigh the possible hazards