Molecular Diagnostics Flashcards
What is the difference between Southern, Northern, and Western blotting?
Southern: probe and target are DNA
Northern: probe is DNA and target is mRNA
Western: used to measure protein
What polymerase is used in PCR and why?
Taq polymerase because it will not denature at a high temp.
What is the difference between PCR and qPCR?
qPCR is used to quantify copy number using a tag that only fluoresces in the presence of the PCR product. Can be used to detect levels of infectious agent or determine levels of gene expression.
What is Restrictive Fragment Length Polymorphism (RFLP) and what is it used for?
A way of DNA fingerprinting by seeing how restriction enzymes cleave an individual’s DNA. Used in forensic analysis, paternity testing, and disease detection.
How is RFLP used in the detection of sickle cell disease?
Normal beta-globulin allele has 3 Ddel restriction sites. Patients with sickle cell only have 2.
What is Variable Number of Tandem Repeats (VNTR) and how does it work?
Used to detect the number of short tandem repeats (STR). Useful in identification and severity of inherited diseases such as Huntington disease, Fragile X syndrome, and Frederich Ataxia.
How is VNTR used to detect Huntington Disease (HD)?
CAG repeats are isolated and ran in PCR. An HD patient will have more repeat sequences than a healthy one.
How are clinically relevant proteins synthesized in vitro?
cDNA of the protein is inserted into expression vectors. Engineered to allow high levels of replication, transcription, and translation to occur in host, leading to large scale production and purification.
How has the uptake of insulin been improved?
Insulin normally has proline at position 28 and lysine at position 29 at C terminus of B chain. Lispro and Aspart alter these positions to make insulin faster acting. Mixing with normal insulin provides longer range of glycemic control.
What is an Enzyme-linked Immunosorbent Assay (ELISA) and how does it work?
A way to test for concentrations of a specific antigen or antibody in biological samples. Uses antibodies conjugated to reactive enzyme. More antigen/antibody = bigger color change.
What is the difference between an Indirect ELISA and a Sandwich ELISA?
Indirect: measures amount of antibody in a sample. Uses antigen-coated well.
Sandwich: measures the amount of an antigen in a sample. Uses antibody-coated well (antigen sandwiched).
How is ELISA used to diagnose HIV?
Uses Indirect ELISA to test for the presence of HIV antibodies in a sample. Needs to be further confirmed using a Western Blot.
How is ELISA used to detect an MI?
Uses a Sandwich ELISA to detect the amount of troponin in a sample. Troponin increases in an acute MI.
How is ELISA used to test for pregnancy?
Uses a Sandwich ELISA do detect hCG in a urine sample.
In a Western Blot, how are proteins separated out prior to the addition of the antibodies?
Via an SDS-PAGE
