Quiz #1 Flashcards
What is the main function of restriction nucleases in bacteria, and how are they protected from them?
Restriction nucleases cut DNA at specific sites.
Bacterial DNA has modifications, often methylations, that restriction nucleases don’t recognize.
Why are enzymes that produce staggered cuts more advantageous over enzymes that produce blunt ends in making recombinant DNA molecules?
It is easier to join DNA with staggered cuts, or “sticky ends” since there are ss, unpaired bases
(Can draw picture)
What reagent allows the visualization of DNA on an agarose gel? And, if you want to visualize and separate very large DNA molecules (>15 Kbs), what technique do you use?
EtBr
Pulsed-field gel electrophoresis
There are two ways (using two different enzymes) to label purified DNA. Name the two enzymes. Which one makes for a stronger probe? Why?
Using DNA polymerase to copy DNA in the presence of nucleotides labeled with 32P or chemically. Makes for a stronger probe bc each nt is labeled
Using polynucleotide kinase to label the 5’ end of each DNA chain with 32P
Northern and Southern blots detect what, respectively? Southern can detect what types of mutations?
Northern blots detect RNA
Southern blots detect DNA
Deletion or insertion
Three common features of an artificially constructed plasmid vectors are? Which one allows for maintaining of the cell?
- Initiation site
- Resistance to antibiotic - maintaining the cell
- Polylinker
What is the main difference between genomic and cDNA library in terms of specificity? Why is that?
Genomic libraries contain the entire genome (introns and exons), making it less specific
cDNA libraries only contain the DNA that is complementary to the mRNA (only exons), making it more specific
What three things do you need in a PCR reaction, in addition to a DNA polymerase?
- Primer
- Nucleotides
- Template strand
What is the advantage of taq polymerase, but what is the problem with taq polymerase?
The advantage of taq polymerase is that it can avoid being deactivated at high temperatures
The disadvantage of taq polymerase is that it lacks 3’ to 5’ exonuclease proofreading activity, resulting in low replication fidelity
What is the main drawback with using primary culture cells?
Primary culture cells die off eventually due to shortening of telomeres
Immortalized cancer cells typically have a mutated version of this gene, which allows for cells to bypass the cell cycle checkpoints. This is important for normal maintenance of cells, i.e. cell division, apoptosis (cell death), etc. What is this gene called?
Oncogenes are the mutated version of protooncogenes
You want to isolate mitochondria from your cells. How do you go about doing this?
Column chromatography. Different spin speeds isolate different elements. A medium spin speed would isolate mitochondria in a pellet.
How does gel-filtration chromatography work?
Based on size. The smaller molecules get trapped inside of the beads and the larger ones travel quickly to the bottom
Give me an example of a recognition tag you can use to make fusion protein.
GST
Concerning SDS-PAGE, the addition of SDS serves what purpose?
SDS is a detergent that denatures the protein, making it linear. It also gives the protein a negative charge.