LEQ: How do we splice new genes into DNA?. 12.1 to 12.7 and 12.18. Recombinant DNA technology. This is a set of lab techniques for combining genes from different sources – even different species – into a single DNA molecule Began with the study of Eschericia coli ( E. coli ).
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12.1 to 12.7 and 12.18
small circular ring of DNA found in prokaryotes and yeast
Important sites of plasmid
1. origin of replication
2. genetic marker (i.e.
3. restriction enzyme
Restriction enzymes have specific recognition sites
Scientists use a specific enzyme to cut out a specific gene and a specific plasmid
The sticky ends of the gene and the plasmid will compliment each other allowing scientists to join the two
Use DNA Ligase to join two DNA types to produce recombinant DNA
Identify a restriction enzyme that will cut out gene of interest and cut open the plasmid
Isolate DNA from 2 sources (making sure the at the plasmid has a genetic marker)
Cut both types of DNA with the same restriction enzyme
Mix the 2 types of DNA to join them through complementary base pairing Add
DNA ligase to bond DNA covalently producing Recombinant DNA
Incubate bacteria at 42 C with calcium chloride; bacteria become competent / permeable - so that the bacteria will take in the plasmid (TRANSFORMATION)
Use a genetic marker to identify bacteria with the recombinant plasmid
Plasmid Libraries and Phage Libraries are created by cutting a genome into fragments
Genome fragments are used to create recombinant DNA
Recombinant DNA is then either stored in a bacterial culture or a phage culture
Transcribe DNA into RNA in the nucleus
RNA splicing occurs – removing introns
Isolate mRNA from the cell and add reverse transcriptase to synthesize a new strand of DNA
The mRNA is digested
Synthesize 2nd complimentary strand using DNA polymerase creating “cDNA”
Contains introns; - must be edited to be get to the DNA that codes for a gene
No introns – the actual DNA that codes for a particular gene
Genetically modified organism – an organism that acquires one or more genes by artificial means (gene may or may not be from a different species)
Transgenic organism – organism that contains a gene from another species
Bacteria are protein factories. E. coli is the primary bacteria used; cheap and easy to maintain cultures and produce proteins (pharmaceutical factories)
Chymosin (used for cheese production), Human Insulin; Human Growth Hormone; Factor VIII; Hepatitis B vaccine; Diagnosis of HIV
The yeast Saccharomyces cerevisiae is commonly used when eukaryotic cells are needed.
Yeast serve as a good vector for human genomic libraries
By genetically modifying the genome of a plant scientists have been able to increase the nutritional value of crops (i.e. “Golden Rice” contains daffodil gene allowing it to produce beta-carotene); scientists have been able to genetically modify plants that are drought resistant, pesticide resistant, larger in size, and that have a longer shelf life.
Recombinant DNA technology is used to add a human gene for a desired human trait (protein) to the genome of a mammal in such a way that the gene’s products, such as antithrombin (protein that prevents blood clots), are secreted in the milk of the animal; Transgenic mammals allow scientists to model human diseases and find treatments to the diseases; Transgenic pigs may serve as human blood and organ donors; Transgenic cattle & fish have been engineered to be larger in size – providing more meat