Molecular Diagnostics Flashcards
Single stranded DNA binds to another strand of DNA or RNA with complementary sequence to form DNA-DNA hybrid or DNA-RNA hybrid.
Hybridization
What is the blotting when both probe and target nucleic acid are DNA? What is it’s purpose?
Southern Blotting
Determine which restriction fragments are associated with a gene
What is the blotting where the probe is single stranded DNA and target is mRNA?
Northern Blotting Measure size and quantities of mRNA molecules (questions about gene expression
What do Western blots target and what is their purpose?
Protein, measure amount of protein or antibody.
What do Eastern blots target and what is their purpose?
Detects post- translational modifications (PTMs) on proteins.
What do you need to conduct a PCR?
dsDNA (patient or pathogen)
Primers that flank each end of DNA sequence of interest in 3’-5’ direction
dNTPs
Taq Polymerases
Thermocycler (denaturing and annealing temp)
Advantage of PCR
Very small amount of template DNA needed, 100-fold amplification from trace amount of DNA
Disadvantage of PCR
Need to know the sequence of the flanking DNA for primer design, error prone, amplification of contaminating DNA
What is the purpose of cell-free PCR
Amplifies isolated DNA regions
Earlier detection of microorganisms: HIV, Lyme disease
Detection of specific genetic mutations
What does qPCR use as an “ingredient” in addition to regular PCR.
A probe which fluoresces only in presence of the PCR product Probe usually a complementary oligo with a fluorescent tag
What is the purpose of qPCR?
Determine levels of gene expression Detect levels of an infectious agent
What is Restriction Fragment Length Polymorphism (RFLP) and how can it be used?
Individual genomes differ by 1 in every 1000 base pairs. Some of these occur at restriction enzyme sites which can be utilized for DNA finger printing in forensic analysis, paternity testing and disease detection.
RFLP can be used for detection of mutants, what is the disease given as an example and how does it work.
Normal β-globulin allele has 3 DdeI restriction sites
Patients with sickle cell only have 2 restriction sites
Followed by electrophoresis and Southern blotting including a β-globulin-specific probe
What is VNTR?
Variable Number of Tandem Repeats
Pattern of short tandem repeats (STR) occurs in genome but varies in individuals These can be isolated by flanking restriction sites r through PCR
Diseases screen in VNTR.
Huntington disease, Fragile X syndrome, Frederich Ataxia.
How are recombinant proteins made?
cDNA of the protein inserted into expression vectors which are put into cell lines.
Examples of large protein production using cDNA
Insulin, growth hormone, erythropoietin clotting factors, vaccines against diseases such as flu, and malaria, viral infections.
How was insulin improved upon?
Normal human insulin has proline at position 28 and lysine at position 29 at C terminus of B chain.
Lispro (Eli Lilly): reversed position of these 2 aa
Insulin aspart (Novo Nordisk): proline 28 replaced by aspartic acid.
Uses of antibodies?
Used as drugs (cancer, rheumatoid arthritis, Crohn’s disease, hepatitis)
And for research purposes
How to make antibodies?
- Single clones of B lymphocytes are fused with a tumor cell to make it immortalized
- Humanization minimizes immunogenicity to prolong half life in patient
What do monoclonal antibodies bind to?
Specific for a single epitope on antigen
Immunological technique which tests for the levels of specific antigen or antibody concentrations in biological samples using a corresponding antibody or antigen.
Elisa
What is measured in direct ELISA, how is this done?
Antigens are measured. 1. Well is coated in antibodies, of which antigen bind to. 2. A secondary antibody is added. 3. Lastly substrate is assessed
What is measured in indirect ELISA, how is this done?
Antibodies are measured 1. Well is coated in antigen, and specific antibody binds. 2.Enzyme linked antibody is added which binds to specific antibody. 3. Substrate is added and converted by enzyme to color
What disease is tested using indirect Elisa?
Why must such diseases be confirmed by Western blotting?
HIV - specific antibodies are developed within 4-6 weeks.
They can produce false positives and false negatives.
What disease is tested for in sandwich Elisa?
Myocardial infarction.
How are pregnancy tests conducted utilizing Elisa?
- Reaction Site
- Test Site
- Control Site
Utilyzes hCG Antigen
1, Reaction site- hCG Antibody binds hCG Antigen
- hCG Antibody complex moves down and bind to immobilized hCG (sandwhich complete = color)
- A non-specific antibody immobilized. Dye gives color regardless of +/- of hCG.
Describe Western Blotting (How is it used? What does it do?)
Used to detect the levels of a target protein in a biological sample consisting of a mixture of proteins
SDS-PAGE = Seperate using charge by size
Transfer proteins from gel to a nitrocellulose membrane
Add Primary
Add Secondary (HRP, Alexa Flor)
Seperate proteins by size through providing electrical feild.
SDS-PAGE
