Cloning Flashcards
Before cloning what is required..
the DNA needs to be cut. up before being inserted into a vector
What cuts the DNA before cloning
Bacterial restriction enzymes
Where does the enzyme EcoRi cut
between G and A nucleotides of each strand
What is the difference between “sticky” end and “overhangs”
Sticky - they stick to complementary sequences
Overhangs - provides cohesive ends for ligation
What are restriction maps
a map of known restriction sites within a sequence of DNA
What is the benefit of a restriction map?
- Provides information on the location of genes
- Restriction maps reflect true physical distance unlike genetic maps
- can help construct physical maps of the entire genome
What can restriction fragments be useful for?
They can be used as molecular markers that reveal a blueprint of an individual genomes
What is gene cloning
the isolation and amplification of a given gene
Steps in cloning a gene
- Cut the plasmid and the DNA with a restriction enzyme that will create identical ends
- Incubate the 2 DNA molecule in the presence of a ligase
- Transform into bacteria to propagate and amplify
What is the benefit of AmpR
can be used to detect the presence of a plasmid backbone - this gene will help make the bacteria grow
Ampicillin
helps to prevent bacterial growth
Can genes stay alive without AmpR
No, because they are unable to grow
What encourages the bacteria to take up plasmids/DNA
the heat shock -
True or False - When a bacteria dies they were able to take up plasmids and AmpR
False
Is bacteria able to grow in the presence of ampicillin and AmpR
Yes, bacteria will be able to go because AmpR can grow in an environment that contains the antibiotic ampicillin.
Purpose of Alpha complementation
to ensure cooling was successful or not
What needs to occur for the cell to become blue?
Blue is an unsuccessful con where DNA was not inserted into the peptides and X-gal was able to be metabolized (B-gal “eats” up X-gal
Beta-gal + X-gal = functional B-gal
What needs to occur for the cell to remain white?
This represents a successful cloning - DNA was inserted into alpha peptides and therefore the X-gal was not able to metabolized. Beta-gal did not cleave X-gal because it had the DNA
No functional Beta-gal
What colour is the cell where there is a alpha complementation versus when there is not
Blue - alpha complementation (unsuccessful cloning) + white is when there isn’t alpha complementation (successful cloning)
True or False - Beta-gal is functional when alpha complementation occurs
Yes true
True or false - in blue colonies DNA was inserted
False, it is with the white colonies that DNA is inserted where there is no functional Beta-gal
What is Southern blotting
a technique used to detect a specific DNA sequence in a blood/tissue sample
Purpose of gel electrophoresis
Through gel electrophoresis molecules in a mixture are separated
What pole do DNA molecules migrate towards during gel electrophoresis?
the positive anode because of its original negative charge
What determines the speed of migration
smaller fragments move easier and large are slower
What makes bands visualized (stain)
Ethidium bromide
What state does the DNA have to be in before proceeding with southern blotting
denatured
Purpose of PCR
Used to quantify DNA in a region of interest between 2 primers
What are the 3 steps of PCR?
- Denaturation
- Annealing
- Extension
What occurs in the step “Denaturation” in PCR?
Temperature is raised to 90 degrees - this breaks the H-bonds between base pairs and releases 2 single strands
What occurs in the step “Annealing” in PCR?
The temperature drops to 40-60 degrees and the short pieces of single stranded nucleotides sequences called primers are used to amplify to target DNA
What occurs in the step “Extension” in PCR?
the temperature is increased to 72 degrees this is ideal for the tax DNA polymerase
Purpose of Taq DNA polymerase
(DNA synthesis) synthesizes the first set of complementary strands by the addition of the four nucleotide triphosphate
What is RFLP
Restriction Fragment Length Polymorphism - A technique that exploits homologous DNA sequences
Benefit of using RFLP
used to distinguish individuals or pinpoint the location of specific genes within a sequence
True or False - Using RFLP technique DNA can be extracted from individuals to map out how a mutation is migrating
True
True or False: When cloning a large fragment it is the same steps as cloning other fragments
False - cannot use plasmids
When cloning large fragment what type of vectors are required
Fosmids + BACs
True or false: vectors in yeast and bacteria have high replicative power
No, false they have low replicative power but are able to carry larger DNA fragments
What is gDNA
Whole-genome -> Genomic library - contains all the genes (introns and exons)
cDNA
Only using exons that will be incorporated into mRNA (coding sequence)
Hybridization
Hybridization allows us to extract and isolate DNA molecules to grow bacterial colony and amplify a desires gene
True or False - with hybridization, you have non-affected bacterial cells and then you need to infect them to see the desired gene amplify?
True
How is hybridization different than finding the clone of interest by using an antibody?
Antibody identifies specific plaques which are fusion proteins bound to the membrane while, hybridization uses bacteriophage to infect bacteria
