DNA Technologies Flashcards
Describe in general the process of PCR.
Polymerase Chain Reaction amplifies large quantities of the desired DNA sequence through the use of successive heating and cooling to denature and re-anneal the DNA, then extension from the primers and replication of the target sequence by DNA polymerase (from a bacteria species called Thermus aquaticus)
What are the practical applications of PCR?
- diagnosis of bacterial and viral infections: primers specific to target sequences of bacteria or viruses are tested on a patient’s tissue or blood sample.
- sex determination of a fetus: primers specific to the Y chromosome on maternal plasma or serum samples
- screen for genetic mutations
- amplify DNA for DNA fingerprinting (RFLP analysis) in paternity testing and forensic investigation
What are restriction endonucleases (enzymes)?
Bacteria have enzymes that can cleave DNA as a defence against viruses. Restriction enzymes recognize specific sequences within the DNA molecule and cleave it within the sequence so that a simple stranded palindromic cut is made. A complementary single-stranded portion of DNA can anneal two different fragments together using ligase
Recombinant DNA
DNA from 2 different sources
Gel electrophoresis
Size separation of DNA fragments. DNA samples are loaded into wells and the surface of the gel is charged with electric current with the (+)ve charge further (so as to attract the (-)ve DNA). Smaller fragments move faster, larger fragments move slower. Size of the unknown fragment is dtm’d by a DNA ladder that contains known fragment sizes.
DNA Sequencing
Too much to put in flashcards. Review notes. Essentially determines the order and compositions of the nucleotide sequence in a sample of DNA by using specific dideoxynucleoside triphosphates (ddNTP’s) which halt the replication of DNA at a specific nitrogenous base pairing,
RFLP analysis
Restriction fragment Length Polymorphism. Mutations that have no adverse effects can occur individually and are inherited. Often these create or remove restriction sites and essentially create different patterns of bands cut by the same restriction sites in different individuals.
Applications of RFLP analysis
Apply to criminal cases, ancestry, paternity cases, etc.
What is the primary application of restriction endonucleases?
Creation of recombinant DNA/genetic engineering. Plasmids are used as vectors for expression of a certain gene once that gene has been cut and pasted into the plasmid by the restriction enzyme. Plasmids replicate by themselves (autonomous) and can be easily absorbed by bacteria (can transmit + express genetic material well) making them good vectors. These bacteria can clone themselves and then the gene of interest can be extracted in greater numbers.
What type of cells can cloning involve?
Stem cells or undifferentiated pluripotent cells are immortal (can divide endlessly) and can differentiate into all types of tissue except placental.
What are induced pluripotent cells?
Adult cells that have already differentiated (somatic) and have been introduced to transcription factors to undifferentiate so as to start over.
What do iPSC’s suffer from?
Enhanced tumorigenesis and teratomas (tumours of different cell types)
Mammary cell
body cell
How was Dolly cloned?
From a somatic cell using the somatic cell nuclear transfer method. Nucleus removed from species A and placed into an enucleated (unfertilized) egg from species B. Electric shocking stimulates division and then the blastocyst formed is inserted into a surrogate.
Gene therapy
Use of genes to treat disease.
