Molecular Diagnostics, Zaidi

Question 1

Hybridization

Answer 1

Used for targeting DNA/RNA
Hybridization of strands

Southern Blot: DNA to DNA, detect unknown DNA
Northern Blot: DNA to RNA, detect RNA

Question 2

Blotting

Answer 2

Southern: detect DNA
Northern: detect RNA
Western: detect proteins (antibodies/antigens)
Eastern: detect Post-translational modifications

Question 3

Polymerase Chain Reaction (PCR)

Answer 3

obtain DNA from a patient/pathogen

normally a small amount and thus needs to amplified via growth by Taq Polymerase

Question 4

Cell Free Cloning (PCR)

Answer 4

amplify isolated DNA regions
early detection of microorganisms: HIV, infections
detection of specific mutations: CF, Huntingtons…

Question 5

Quantitative PCR (qPCR)

Answer 5

real time PCR
quantify copy number of specific gene in 2+ samples in real time
detect levels of infectious agent
determine gene expression (incr/decrease in expression)

Cancer graph: cancer has higher amounts of target DNA sequence

Question 6

Restriction Fragment Length Polymorphism (RLFP)

Answer 6

DNA finger printing
forensic analysis, paternity tests, disease detection

ex: crime scene, suspects vs sample found
ex: detect mutations, sickle cell, restriction sites in beta-globulin allele, 3 cuts vs 2 (sickle)

follow up with electrophoresis and Southern

Question 7

Variable number of tandem repeats (VNTR)

Answer 7

short tandem repeats occurs in genome

identification/severity of inherited diseases
Huntingtons (look for number of repeats in sequence of CAG’s), Fragile X, …

Question 8

Recombinant Proteins

Answer 8

large scale productions

ex: insulin from bacteria using plasmids , antibodies from mice

Question 9

Insulin

Answer 9

normal: proline at 28, lysine at 29

Lispro: switched, proline at 29, lysine at 28
Insulin Asp: aspartatic acid switches with proline at 28

These are faster acting and have better glyc. control

Question 10

Enzyme linked immunosorbent assay (ELISA)

Answer 10

tests for levels of antigen or antibody concentrations

ex: indirect vs. sandwich

Question 11

Indirect ELISA

Answer 11

measures amount of antibody in sample

start with antigen coated well, add antibody, E-linked antibody next

ex: HIV (need to use Western Blot as well)
detect HIV p24

Question 12

Sandwich ELISA

Answer 12

measures amount of antigen in sample

start with antibody coats well, antigen binds, second antibody creates sandwich with antigen in middle

ex: pregnancy test, Myocardial infarction

Question 13

Western Blotting

Answer 13

detect levels of target proteins in sample

use SDS-PAGE to separate proteins on gel
protein migration, add primary then secondary antibodies