Gene Isolation and Manipulation Flashcards
DNA Technology -
The collective techniques for obtaining, amplifying and maniuplating fragments
What is one of the foundational examples of biotech?
Recombinant human insulin in large quantites
Genetic engineering -
The application of DNA tech to specific biological, medical or, agricultural problems
Genomics -
Extension of DNA technology to the global analysis of the nucleic acids present 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
Restriction enzymes digest DNA at specific sequences
- Cut DNA at specific motifs
PCR reactions amplify specific DNA sequences
Whats the general process?
A series of Denaturing, annealing, and extending events
cDNA can be made by reverse transcription which makes?
makes copies of DNA without introns
What is recombinant DNA?
3 step process?
How do you provide directionality?
You take an insert (target gene) and place it in a vector
1. Use restriction enzymes to produce sticky ends on insert and vector
2. Anneal the insert with the vector
4. DNA ligase to seal nicks
Use more than one restriction enzyme to ensure directionality
Plasmids have additional features built in. What does PUC18 enable?
Enables Blue/white screening for cloned DNA to see which colonies have inserts and which dont.
Blue colony means?
Cleavage present, no DNA insert present
White colony means?
No cleavage, DNA insert present
What does the plasmid pET enable?
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?
Assemble 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
What are the 5 main in VITRO methods used to detect and quantify specific DNA regions, RNAs and proteins?
DNA- Southern Blot and Polymerase Chain Rxn
RNA - Northern Blot and PCR
Protein - Western Blot
What is the probe for Southern blot and Western Blot
DNA or RNA fragment
What is the probe for PCR?
DNA primers
What is the probe for Reverse transcription - PCR (RT-PCR)?
DNA primers
What is the probe for Western blot?
Antibody
What are the main in VIVO methods used to detect and quantify specific DNA regions, RNAs and proteins?
1.FISH- Fluorescence in situ hybridization
2. In situ hybridization
3. Immunofluoresence
What is the probe for FISH and In situ hybridization?
DNA or RNA Fragment
What is the probe from Immunofluoresence?
Antibody
What can probes be used to identify?
Probes can identify genomic DNA regions containina a gene of interest
What can agarose gel electrophoresis do?
Separates DNA fragments based on size
What is the main point of using a restriction enzyme?
To hybrizide a piece of DNA with some type of plasmid so you can move it into E.Coli or some type of cellular organism.
Fusing whatever youre cutting with a plasmid.
What if you want to work with mRNA so that there are no introns present in your template?
aka RNA Pol II transcribed genes
cDNA can be made by reverse transcription
Once you have amplified DNA from PCR what do you need to do?
Put it into a vector so that it can be maintained in cells by producing recombinant DNA
How do you produce recombinant DNA from amplifed DNA?
Cut the DNA (insert) and vector with restriction enzymes to produce sticky ends that will anneal with each other
What does the His-tag do?
Makes it easier to fish out the protein of interest for synthesis
What vector can handle a 35-45kb size DNA insert?
Fosmid
What vector can handle a 100-200kb sized DNA insert?
BAC- Bacterial Artificial Chromosome
What vectors enable big inserts?
Fosmids and BACs
Why would you need to add tails fo cDNAs using adapters?
So that you can produce a site that a resctriction enzyme can turn into a sticky end for hybrisization with a vector
