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Flashcards in Molecular Diagnostics, Zaidi Deck (13)
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1
Q

Hybridization

A

Used for targeting DNA/RNA
Hybridization of strands

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

2
Q

Blotting

A

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

3
Q

Polymerase Chain Reaction (PCR)

A

obtain DNA from a patient/pathogen

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

4
Q

Cell Free Cloning (PCR)

A

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

5
Q

Quantitative PCR (qPCR)

A

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

6
Q

Restriction Fragment Length Polymorphism (RLFP)

A

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

7
Q

Variable number of tandem repeats (VNTR)

A

short tandem repeats occurs in genome

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

8
Q

Recombinant Proteins

A

large scale productions

ex: insulin from bacteria using plasmids , antibodies from mice

9
Q

Insulin

A

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

10
Q

Enzyme linked immunosorbent assay (ELISA)

A

tests for levels of antigen or antibody concentrations

ex: indirect vs. sandwich

11
Q

Indirect ELISA

A

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

12
Q

Sandwich ELISA

A

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

13
Q

Western Blotting

A

detect levels of target proteins in sample

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