Manipulating Genomes Flashcards
What is the PCR?
method for amplifying DNA fragments rapidly and effectively
What are primers?
Short sequences of nucleotides that attach to the start and end of a DNA fragment to be copied
what components are required in PCR?
DNA fragment
Primers
Taq polymerase
Free nucleotides
Thermocycler
What is Taq polymerase?
A type of DNA polymerase from thermophilic bacteria
can withstand high temperatures without denaturing
What is stage 1 of PCR?
Separation
Temp: 95 degrees celsius
DNA is heated to separate the 2 strands by breaking the hydrogen bonds
What is stage 2 of PCR?
Annealing (adding primers)
Temp: 55 degrees Celsius
Primers attach to the specific start and end points of the separated DNA strands
What is stage 3 of PCR?
DNA synthesis (extension)
Temp: 72 degrees Celsius
Taq polymerase adds free nucleotides to the target strands to form a complete copy of the strand
Advantages of the PCR
Rapid speed
Precision
Only small samples of DNA needed
No cells needed
What is gel electrophoresis?
technique used to separate DNA based on size using an electric current applied to an agarose gel matrix
How is gel electrophoresis set up?
Insert a gel tray with solid agarose gel into a gel tank
Ensure walls are close to negative electrode to position gel correctly
Pour buffer solution over gel until submerged
How are samples loaded in electrophoresis
Mix DNA samples with a dye to make them visible
Micropipette equal volumes into the wells
What happens during electrophoresis
A voltage is applied across the Gel
Fragments of DNA move toward the positive electrode
Smaller fragments travel faster
What are the key stages of gene transfer?
Desired gene is identified and isolated
Multiple copies made using PCR
The genes is inserted into vector
Vector delivers gene into cells in different organism
Cells with new gene are identified using marker genes
Cells with new genes are cloned
How are DNA fragments produced
mRNA is extracted from cells
mRNA is reverse transcribed by reverse transcriptase and free nucleotides to form cDNA
Free floating nucleotides and DNA polymerase are used to form the other strand of cDNA
What are restriction enzymes
recognize and cut DNA at specific palindromic nucleotide sequences to isolate gene fragments
palindromic nucleotides read the same forward and backward
How do restriction enzymes work?
DNA incubated with chosen restriction enzyme
Restriction enzymes identify palindromic sequences and cut DNA if their recognition sequence is present
Recognition sequences are at the beginning and end of desired DNA fragment
Enzymes cut target gene fragment out by hydrolysis
What are sticky ends?
Short overhanging sequences of unpaired bases that can bind to other DNA fragments
How are DNA fragments inserted into vectors
Vector is cut open at specific site using restriction enzyme - creates sticky ends
Same restriction enzyme is used to cut target DNA fragment to make complementary sticky ends
DNA ligase forms phosphodiester bonds, joining the sticky ends of the vector and DNA fragment
Recombinant DNA formed
How is recombinant DNA transferred into host cell using plasma vectors?
Host cells are treated to enhance uptake of plasmids that have recombinant DNA
Adding calcium ions and temperature shifts increases membrane permeability to plasmids
Electroporation uses an electrical current to make bacterial membranes more porous
helps plasmids enter
How is recombinant DNA transferred into host cell using bacteriophage vectors?
Bacteriophages inject their DNA into host bacterial cells during infection
Recombinant DNA is injected into host cell
How can transformed host cells be identified?
Marker genes indicate which host cells have recombinant DNA
Inserted into vectors alongside target genes
transformed cells are cultivated on selective agar plates
Only transformed cells are marked
Transformed cells are cultured to be mass produced
What are the different types of marker genes?
Marker gene for a specific trait
Marker gene that is visible under UV light
Marker gene coding for a specific enzyme that changes the colour of a specific substrate
