Molecular Diagnostics Flashcards
What are the techniques used to detect infectious agents and diagnose inherited disorders
- Hybridization
2. Polymerase Chain Reaction (PCR)
What must be known for hybridization and PCR to move forward
You must have to know the sequence of the pathogen or the sequence
What is hybridization used for?
Used to detect target DNA (southern blot) or RNA (northern blot) in a sample
In hybridization, what kind of sequence do you need for a single-stranded DNA to bind to another DNA or RNA strand?
You need a complementary sequence to for the DNA-DNA (southern) hybrid or the DNA-RNA (northern) hybrid
What is meant by “blotting”
When the target DNA is converted to a single strand DNA, it is then IMMOBILIZED onto a membrane
What’s the difference between Southern and Northern blotting
Southern: both probe and target sequence is DNA
Northern: probe is single-stranded DNA and target is mRNA
What is the purpose of blotting techniques
To detect and visualize specific biomolecules present in a mixture. Why you must immobilize onto a membrane.
What is the purpose of a Southern blot
Determine which restriction fragments are associated with a gene
What is the purpose of a Northern blot
Measure the size and quantity of mRNA
What is the purpose of a Western blot
Measures amount of protein or antibody
What is the purpose of Eastern blot
Detects post-translational modifications (PTMs) on proteins
SNOWDROP
S-southern D-DNA
N-northern R-RNA
O and O nothing
W- western P-protein
What are the ingredients for PCR?
Double-stranded DNA Primers dNTPs Taq polymerase (heat resistant) Thermocycler
What are the steps of PCR?
FIRST CYCLE 1. Heat to separate DNA strands 2. Cool to anneal primers 3. DNA synthesis (w/ dNTPs and DNA polymerase) produces 2 double-stranded DNA molecules
SECOND CYCLE
1. Heat the 2 double-stranded strands and cool to anneal primers
2. DNA synthesis
produces 4 double-stranded DNA molecules
THIRD CYCLE
1. Heat the 4 double-stranded strands and cool to anneal primers
2. DNA synthesis
produces 8 double-stranded DNA molecules
What is quantitative PCR used for?
Lets you quantify how many copies of a specific gene are present. Used to detect levels of an infectious agent and determine levels of gene expression.
What 2 processes detect variations in DNA sequences?
- Restriction Fragment Length Polymorphism (RFLP)
2. Variable number of tandem repeats (VNTR)
When is RFLP used?
DNA fingerprinting, forensic analysis, paternity testing, disease detection
How many restriction sites does a normal-globulin allele have?
3 Ddel restriction sites. Will produce 2 on a membrane.
How many restriction sites dows a sickle cell pt have on their B-globulin allele?
2 restriction sites. Will produce 1 on a membrane.
What can VNTR help diagnose?
Useful in identification and severity of inherited diseases.
Huntington, Fragile X, Frederich Ataxia
In order to diagnose Huntington, what must you know before you carry out VNTR?
Must know the repeats and make primers for the repeats
In recombinant proteins, what is inserted into the expression factors?
cDNA of the protein is inserted into the expression factors
What are some examples of recombinant proteins produced?
Insulin Growth hormone Erythropoietin Clotting factors Vaccines against flu, malaria, etc
How does recombinant protein manufacturing work?
- Take a plasmid from a bacterium and whatever gene from a human.
- Put that gene into the plasmid so that they’re “mixed”
- Put the gene+plasmid (recombinant DNA) into a bacterial cell
- Put into a fermentation tank
- The recombinant bacteria multiply and produce whatever protein from the gene you selected for at the beginning (i.e. insulin)
What are some “improved” forms of insulin
Lispro (reverse position of proline and lysine)
Insulin aspart (proline 28 replaced by aspartate
*both of these are faster acting and better absorbed
What position are proline and lysine at in normal insulin
Proline at 28
Lysine at 29
At C terminus od B chain
Apciximab
Inhibits platelet aggregation
Apciximab=Aggregation
Baciliximab
Prevents rejection of transplanted kidney
Kids are BAD (baciliximab)
Infliximab
Treats autoimmune disease
Inflix=autoIMMUNE
Cetuximab
Treats metastatic colorectal cancer
Cetux=colorectal cancer
Retuximab
Treats lymphomas and leukemia
LLR like the LIRR
What does the ELISA (enzyme-linked immunosorbent assay) test for?
Tests for levels of a specific antigen or antibody concentrations in a biological sample using a corresponding antigen or antibody
What does an indirect ELISA test for?
Measures the amount of antibody in a sample
What does a sandwich ELISA test for?
Measures the amount of antigen in a sample
What can you diagnose w/ ELISA?
HIV (indirect ELISA)
MI (Sandwich ELISA)
Pregnancy (Sandwich ELISA)
What is an SDS-PAGE?
A type of western blot (immunoblotting). SDS-PAGE separates out the proteins on a gel by applying electrical field. Smaller proteins move faster than larger ones
What are the steps in an SDS-PAGE?
- Load protein samples onto SDS polyacrylamide gel and apply an electrical field
- Transfer protein to the membrane
- Add primary antibody that binds to target protein. Wash.
- Add tagged secondary antibody that binds to the primary antibody. Wash.
- Use visualization agent to detect the secondary antibody signal
What test would you use to confirm HIV?
Western blot. Would see HIV p24 antigen even before antibodies are formed. Other antigens include gp160, gp120, p66, p55, etc.
