Molecular Diagnostics Flashcards
What is the probe in Southern Blotting?
DNA
SNOW
DR P
What is the target in Southern Blotting?
DNA
both are DNA
What is the target in Northern Blotting?
mRNA
Snow drop
What is the probe in Northern Blotting?
single stranded DNA
What is the target in western blotting?
Protein
What is the basic sequence of PCR?
put in your dna, primers, and enzymes denature DNA by heating cool it down to allow primers to anneal Add dNTPs Taq pol will extend primers
What is the disadvantage of PCR?
need to know the sequence you are looking to amplify
What is the point of PCR?
to amplify a sequence or see if it is there at all
What does the number of cycles mean in qPCR?
high number of cycles = little protein
small number of cycles = a lot of protein
What is restriction fragment length polymorphism (RFLP) and what is it used for?
Detects differences in genomes by comparing the lengths of fragments after dna is cut with endonucleases
Used for forensics, paternity testing and disease detection
How many restriction sites does a normal beta globulin allele have?
3 –> creates 2 pieces of gene = 2 bands on normal blot
How many restriction sites does a sickle cell beta globulin allele have?
2 –> only one piece of dna = 1 band on homozygous pt; 3 on heterozygous (1 copy of normal, 1 copy of abnormal)
What do you use variable number of tandem repeats (VNTR) for?
identification and severity of inherited diseases
Huntington’s
What is a plasmid vector?
Circle of DNA from bacteria; where you put your human gene to produce something
What is recombinant dna?
Plasmid vector with human dna in it
What type of cells are taken from an animal to produce monoclonal antibodies?
spleen cells
What are spleen cells fused with to make immortal cells for monoclonal antibody production?
myeloma cells –> hybridoma
What does an indirect ELISA measure?
a specific antibody
antigen coated well –> Ab binds –> secondary Ab w/ enzyme binds –> color change
What does a sandwich ELISA measure?
A specific protein/antigen
Antibody coated wells –> Antigen binds –> secondary Ab w/ enzyme binds –> color change
What technique is used to first detect HIV?
What type of molecule is it testing for?
ELISA
Testing for antibodies produced 4-6 weeks after infection, so use indirect ELISA
What technique is used to detect an MI?
looking for troponin –> Use sandwich ELISA bc looking for an antigen
Which type of ELISA is used in pregnancy tests?
looking for an antigen (hCG) –> use sandwich ELISA
What technique is used in the confirmation of HIV?
Western blotting
What is the general process of western blotting?
SDS-PAGE to separate proteins –> transfer to nitrocellulose membrane –> and 1 antibody –> and 2 antibody w/ enzyme tag –> and substrate –> color that is proportional to amount of protein
