10/20 Quiz Flashcards
DNA Technology -
The collective techniques for obtaining, amplifying and maniuplating fragments
Genetic engineering -
The application of DNA tech to specific biological, medical or, agricultural problems
Genomics -
the extension of DNA technology to the global analysis of the nucleic acids presnt in a nucleus, cell, organism or group of related species
How do you make recombinant insulin?
4
- Plasmid with human insulin gene is put into bacteria.
- Bacteria is put into fermentation tank and produces insulin
- Harvest and purify insulin produced
- purified insulin is turned into medicine
Southern/Northern blot steps
5
- cleave DNA
- gel electrophoresis
- Gel gets filtered
- expose to radiolabeled complementary DNA
- Expose to xray
What do restriction enzymes do
Enzymes digest DNA at specific sequences
- Cut DNA at specific motifs
PCR reactions amplify specific DNA sequences
Whats the general process?
Denature, anneal, extend
cDNA cane be made by reverse transcription which makes?
makes copies of DNA without introns
What is recombinant DNA?
4 step process?
How do you provide directionality?
You take an insert (target gene) and place it in a vector
1. cleave vector
2. insert gene
3. anneal
4. DNA ligase
Cut 2 ends for directionality
PUC18 enables?
Blue/white screening for cloned DNA
Blue colony means?
Cleavage present, no DNA insert present
White colony means?
No cleavage, DNA insert present
pET enables?
ENables induction of cloned gene and addition of his-tag
3 ways recmobinant DNA can be delivered into bacterial cells?
Plasmids and BACs
Fosmids
Bacteriophage vectors
2 ways you can make large DNA constructs?
Assamble from multiple restriction products
Gibson assembly
Large DNA molecules can be assembled by transforming overlapping constructs into yeast using…
Homologous recombination
what does dNTP have that ddNTP doesnt?
dNTP has a hydroxyl goup at the 3’ position.
ddNTP will stop DNA polymerase from advancing
Where does ddNTP stop the DNA polymerase?
ddATP-Adenine
ddTTP- Thymine
ddGTP- Guanine
ddCTP- Cytosine
5 methods for delivering transgenes
- Lipid vesicle - mRNA vaccine
- electroporation - stress cell till membrane opens up
- biolistic delivery - DNA coated particles shot into cells
- micro injection - bypass membrane with a needle and inject
- virus infection
What do you need to happen for homologous recombination?
You need double crossover
Where do you inject transgene in C. elegans
in the synctial region. Some eggs will eventually have the transgene
Drawback of chimeras?
Recovering homozygous mutants from chimeras takes multiple generations
What does CRISPR/Cas9 do?
Complexes with guide RNAs to catalyze double stranded breaks in DNA sequences
What are teh two ways DNA can be repaired after a DSB?
Homologous recombination
Nonhomologous end joining
3 types of nonhomologous end joining? NHEJ
Insertion
Substitution
Deletion
How does a repressor recognize the gene to repress?
The operator, a specific DNA sequence
How does the cell decide whether it should go for
lactose?
Glucose levels: high or low
When is cAMP produced?
Low glucose levels
When is cAMP produced?
Low glucose levels
