4. Molecular Diagnostics Flashcards
What two types of diagnostic tests can be used to detect infectious agents and diagnose inhereted disorders?
Hybridization and PCR
DNA-DNA Hybridization is also called what?
Southern Blot
Jokes about cousins marrying in the south. DNA-DNA joining.
What does Hybridization allow you to do, in simple terms?
Figure out, in the presence of other DNA, if a specific sequence of DNA is present, and if so, how much.
What are the basic steps of Northern / Southern Blotting?
Isolate single stranded, enzyme-cut DNA
Use electrophoresis
Tranfer the “sorted” DNA to a membrane
Wash the probe (complimentary sequence) over the fixed DNA
Read where the probe stuck to the target DNA
What does Western Blotting look for?
The amount of a protien or antibody
What is the point of PCR?
What all ingredients do you need?
Amplifies a DNA sample.
DNA Sample
Short Primers (To prime for new DNA)
Heat-Stable DNA pol (to synthesize new DNA)
dATP, dGTP, dCTP, dTTP (building blocks for new DNA)
Thermocycler (for annealing and denaturing)
What does qPCR measure?
What do higher numbers mean?
The amount of a target DNA, measurement given in cycles necessary to reach an arbitrary threshold.
Less of the measured DNA is present, since it took more cycles of doubling to reach the threshold.
What are the two methods for determining differences in a DNA sequence?
Restriction Fragment Length Polymorphism
Variable Number of Tandem Repeats
How do restriction enzymes help differentiate between DNA sequences, say between one person to another?
What technique takes advantage of this phenominon?
For a given set of restriction enzymes, the same region of DNA on two people might be cleaved in one case, but not in another. this shows that they are different by revealing DNA strands of different length.
Restriction Fragment Length Polymorphism (RFLP)
What does Variable Number of Tandem Repeats help us identify?
Diagnosis of and severity of inherited diseases.
What are the characteristics of polyclonal antibodies?
They are antibodies that affect multiple epitopes of one antigen
What are the characteristics of monoclonal antibodies?
The only attach to one specific epitope for a given antigen
Which type of ELISA measures the amount of an antigen using antibodies?
Sandwich ELISA
I think it’s called Sandwich ELISA because from bottom to top it goes Antibody-Antigen-Tagged Antibody; like an antigen sandwich.
What kind of ELISA measures the amount of an antibody using an antigen?
Indirect ELISA
What is the difference between an ELISA and a Western Blot?
ELISA is more quantitative, Western Blot is more visual. Also, Western Blot can only test for antigens (like sandwich ELISA).
