Flashcards in 8.9.16 Lecture Deck (48)
What are restriction enzymes?
Restriction enzymes cleave double stranded DNA at specific nucleotide sequences.
The nucleotide sequences at which restriction enzymes cleave are often ___.
Restriction enzymes can cut in two ways - describe these.
Symmetrically (creates blunt ends) and asymmetrically (creates sticky ends, which can base pair)
DNA fragments can be ___ to produce new combinations.
___ can join DNA fragments cut with the same restriction enzymes.
Describe the process of creating recombinant DNA.
1. Restriction enzyme cuts DNA at the recognition sequence, creating 2 sticky ends.
2. DNA fragment from another source is added and fragments stick together by base pairing (annealing).
3. DNA ligase covalently seals the cut sites, creating recombinant DNA.
A DNA fragment can be inserted into a bacterial ___ with DNA ligase.
What is a plasmid?
Double-stranded DNA molecule that is separate from and can be replicated independently of chromosomal DNA.
Describe the process of incorporating DNA into a plasmid.
1. Cut open the plasmid with a restriction nuclease
2. Mix the cut plasmid with the DNA fragment to be cloned. Note that this fragment is prepared with the same restriction nuclease.
3. DNA ligase and ATP are added.
4. The ends base pair and DNA ligase seals the nicks in the backbone with covalent bonds.
Describe the process of amplification with plasmids and bacteria.
1. Insert the fragment of DNA into the plasmid vector
2. The recombinant plasmid is introduced into a bacterium through transformation.
3. The bacterium replicates millions of times, creating many copies of plasmid.
4. Lyse the bacteria and collect the purified plasmid.
Recombinant DNA can be used to produce clinically relevant proteins. Describe how this works and give two examples.
Some plasmids contain the appropriate genetic elements to force transcription and translation of the inserted DNA fragment. Cells can be forced to produce large quantities of proteins. Examples include insulin, human growth hormone, taxol, cellulose, factor VIII.
How does gel electrophoresis work?
1. Cut wells in the gel.
2. Submerge the gel in buffer (maintains pH, contains necessary ions)
3. Apply a current
4. DNA (negatively charged) runs toward the anode (positive end)
Gel electrophoresis can separate DNA by size. Smaller molecules run ___ down the gel.
What type of gel is used for short DNA molecules (
What type of gel is used for large DNA molecules (>50,000 base pairs)?
Pulsed-field agarose-gel electrophoresis
Complementary nucleic acid strands can bind to each other through nucleic acid ___.
How is DNA denatured?
Heat or high pH (alkali solution)
What is Southern blotting?
Combines electrophoresis and hybridization to detect a specific DNA molecule within a complex mixture.
How does Northern blotting differ from Southern blotting?
Northern blotting is for detecting RNA sequences
What are probes?
Single-stranded DNA molecules used to detect complementary sequences; carry radioactive or chemical markers to facilitate detection
Describe the Southern Blot analysis process.
1. Cleave DNA with a restriction enzyme
2. Run unlabeled DNA on agarose gel to separate by size.
3. Separated nucleic acids are blotted onto nitrocellulose paper by suction of buffer through gel and paper (transferred to a membrane)
4. Remove nitrocellulose paper with bound nucleic acids
5. Radiolabeled probes are hybridized to separated DNA
6. Labeled probes hybridized to complementary DNA bands are visualized.
What is RFLP?
Restriction Fragment Length Polymorphism
Sickle-cell disease can be detected through ___.
In sickle-cell disease, Glu mutates to Val, which destroys ____.
A restriction enzyme cut site
How can RFLP be used to determine the presence of sickle-cell mutation?
Because the restriction enzyme cut site is removed, the mutated gene will be longer than the unmutated gene.
What does in situ hybridization do?
Locates specific DNAs within cells and genes on chromosomes.
Describe the process of FISH (Fluorescent in situ hybridization).
1. Denature DNA to create single-stranded DNA.
2. Bind fluorescent to complementary sequences
3. Allows for visualization of multiple probes simultaneously
FISH is utilized in the clinical lab for the diagnosis of ___.
Her2 positive breast cancer
What is PCR?
Polymerase Chain Reaction; process of amplifying DNA through repeated rounds of synthesis in vitro