pack 4 Flashcards
(28 cards)
What do the southern northern and western blots measure
Southern is for DNA
northern is for RNA
western is for proteins
What are the steps of southern blot?
You extract the DNA
add restriction enzymes to cleave the DNA
run through gel electropherosis
put the filter on top
put paper towels so it can suck it up
add the probes in a bag
What are the two different types of detection that are used for the southern blot
Radioactive and chromogenic
What are some applications of southern blot?
Cloning/verfication of cloning
Forensic parentage testing
Detect DNA mutations that cause the loss or gain of restriction sites
What does a mutation that causes a gain or loss of a restriction site do to an electropherosis results sheet like the one used in southen blot?
It changes the place of the lines bc:
Loss of restriction site –> Dna will not be cleaved –> it will be heavy and will stay behind
Gain of restriction site: DNA will be cleaved more than normal so it will be light so the line will be further away from normal
aa in this case lost a restriction site
What is the difference between dot blot and southern blot?
Dot blot: Used to screen a huge number of samples
tells you only whether a specific mutation is present or not and not its site
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Southern blot: Not used for screening
Used on small samples
Used to tell you whether or not the mutation is present and it size (presence or absence of restriction site)
What are the 4 types of probes?
What is the normal amount of red and green in FISH?
2 red
2 green
What is the job of Sodium dodecylsulfate (SDS) in gel electrophoresis?
It wraps around the molecules and turns them all negative
What type of blotting analyzes gene expression?
Northern blotting
What type of electropherosis does western blotting use? (horizontal, vertical, etc?)
SDS PAGE (vertical)
What is the probe that we use for western blotting?
antibody
How does western blotting work?
You have your main antibody that binds to the protein
Then a second antibody that has a a flourscent enzyme binds directly to the first one (this is the difference between it and elisa)
almost like elisa
What are the two types of elisa?
Indirect and sandwich ELISA
How does indirect ELISA work?
A plate is coated with an antigen (difference)
The antibody from the patients sample will bind to the anitgen
A secondary antibody with an enzyme is added and it will bind to the first antibody
If a substrate is added, a signal will be immited
What does indirect ELISA detect?
antibodies in a patients serum
How does sandwich ELISA work?
Coat the plate with antibody (difference)
Add the sample from the patient
The antigen will bind to the first antibody
A second antibody with a flourscent enzyme will bind to the antigen as well (difference)
If a substrate is added, a signal will be emitted
How is ELISA and western blotting used togther to ensure accuracy of results?
ELISA is first used bc it is fast and inexpensive
If the patient is positve, they are retested using western blotting to ensure accuracy as elisa has a 20% chance of false positives
What is polymorphisim?
A physiological variation in a genetic sequence present in at least 1% of the population
What are SNPs?
Single nulceotide polymorphisim
What can SNPs do in relation to restriction endonucleases?
they can add or remove restriction sites
Explain in this photo why the normal person has only one line while there are 3 fragments?
bc of the fact that the probe got attached to the middle segment only
What is VNTR?
Variable number tandem repeats
It is the insertion or deletion of 2-4 nucleotides
How is VNTR studied?
Southern blots or PCR
