Molecular Diagnostics Flashcards
What are the techniques used to diagnose infectious diseases?
1) Hybridization
2) Polymerase Chain Reaction (PCR)
What is Hybridization?
1) Single-stranded DNA (called a probe) binds to either DNA or RNA with a complementary sequence to form a hybrid
- Allows us to detect and quantify if a specific sequence is present
- If there is a match between the sequence and the complement, there will be fluorescence
What are the various blotting techniques and what do they allow us to determine?
Uses gel electrophoresis
1) Southern (DNA) - what restriction fragments are associated with a gene
2) Northern (RNA) - Size and quantities of mRNA molecules
3) Western (protein) - measure amount of protein or antibodies
4) Eastern (lipid, carb) - Detects PTMs
What is PCR, it’s steps and advantages/disadvantages?
-Polymerase Chain Reaction - allows for DNA amplification
1) DNA obtained from sample
2) DNA denatured to separate strands
3) Primers flank each strand
4) Taq Polymerase synthesizes DNA copy, doubles DNA in each sequence
Ad: small amount of DNA needed
Dis: need to know DNA flanking sequence
What does Cell-Free Cloning (PCR) allow us to do?
- Amplify isolated DNA regions
- Early detection of microorganisms and specific genetic mutations
Explain Quantitative PCR (qPCR)
- Used to get a specific number of copies of a specific gene in real-time (i.e. can see if cancer has more of a specific gene than a normal cell)
- Probe with fluorescence that goes off only when PCR product is present
1) Detects levels of infectious gene
2) Determines levels of gene expression
What are Restriction Fragment Length Polymorphisms (RFLP)?
- Technique that allows one to identify unique DNA sequences
- Uses specific restriction enzymes to cut sequence and compare homologous strans
What is RFLP used for?
- Forensics, paternity
- Disease identification – Sickle cell has two restriction sites = 1 fragment
(Think R the Father)
What are Variable Number of Tandem Repeats (VNTR) and what are the uses?
- repeated segments of the genome that are specific in individuals
- isolated any flanking the sequence through 1) restriction sites and 2) PCR
- IDENTIFYING AND FIGURING OUT DISEASE SEVERITY (Huntington’s, Fragile X, Frederich Ataxia)
-Process: Fragments separates on gel
What can VNTRs help us identify?
1) Prenatal diagnoses
2) Newborn Screening
3) Carriers of disease
What are Recombinant Proteins?
- Allow for large scale production of proteins
- Insulin, growth hormone, erythropoietin\, clotting factors, vaccines against diseases
Recombinant protein production steps
1) cDNA inserted into expression vectors
2) Designed to have high replication levels
3) Large scale production and purification
Insulin improvements
Lispro and Aspart — made changes in proline and lysine positions to improve:
1) speed of insulin 2) absorption
-Mixing with normal insulin provides longer range
Briefly explain antibody production
1) Put antibody within spleen cell of a rat
2) Have B cells clone itself along with a tumor cell to keep proliferating
3) Monoclonal antibodies extracted
Monoclonal vs. Polyclonal antibodies
Monoclonal - targets one specific part of the molecule
Polyclonal - targets various parts of the molecule
Abciximab
Abs that inhibits platelet aggregation (A for aggregation of platelets
Baciliximab
Abs that prevents rejection of translated kidneys (B for send Back)
Cetuximab
Treats metastatic colorectal cancer (C for colorectal)
Infliximab
Treats autoimmune disease (I for immune)
Retuximab
Treats lymphomas
What is Enzyme-linked Immunosorbent Assay?
- ELISA
- Test for antigen or antibody concentrations (western blot can only detect the presence)
How does ELISA work?
1) Insert antibody or antigen into host with a reactive (color-changing) enzyme
2) Binding competition between antigen you’re searching for a antigen-conjugate occurs.
3) If the antigen is present, a change in color is observed — color hue determines relative concentration (since there I competition with conjugate)
Rate of color formation is dependent on the amount of the specific antibody
What is the difference between Indirect ELISA and Sandwich ELISA?
Indirect - measures amount of an antibody in the sample (antigen starts, coated on the well bottom)
Sandwich - measures the amount of the antigen in the sample (antigen is “sandwiched” between two antibodies)
What can ELISA help diagnose?
1) HIV (need to confirm with western blot since false negatives and positive are sometimes common
2) Heart Attack (MI) – Troponin I, Troponin C increase following MI
3) Pregnancy – free antibody on stick traps substrate (human chorionic gonadotropin -hCG) and sandwiches it a fixed antibody, which releases dye substrate
Western Blot
- aka Immunoblotting
- Detects target protein concentration
- Confirms HIV results
Western Blot steps
1) Proteins separated based on weight in SDS-PAGE via electrical field
2) Proteins moved to NITROCELLULOSE
3) Primary and secondary antibodies are added
4) Enzyme tag on secondary antibody integrates with added substrate, GIVES COLOR!!
