molecular diagnostics Flashcards
What is hybridization? what does it entail?
Used to detect and quantify target DNA or RNA
Must know sequence of pathogen
Single stranded DNA binds to another DNA or RNA to form hybrid
Target DNA first converted to SSDNA then immobilized
Southern blot used for what?
DNA & nucleic acids
Northern blot: used for what?
RNA size and quantities
western blot: used for what?
proteins
Eastern blot used for what?
PTM
PCR, what does it entail?
Double stranded DNA obtained from source, make many copies of dna
Denatured at high temp
Primers designed to complement source DNA
Allowed to anneal (binding to DNA)
Addition of dNTPs (dAtp,dTtp,dCtp,dGtp)
Uses Taq Polymerase
DNA doubles after each cycle
positives and negatives of pcr?
Advantages: very small amount of template DNA needed, quick amplification of DNA
Disadvantages: need to know sequence of the flanking DNA or primer design, error prone, amplification of contaminating DNA
RT-qPCR
used to quantify copy number of a specific gene in two or more samples in real time
Detects levels of an infectious agent
Determine levels of gene expression
In addition to primers, this techniques uses a fluorescent probe only in presence of PCR product
-Probe usually a compliment with a fluorescent tag
what are variations in DNA used for?
Prenatal diagnosis (amniocentesis) Newborn screening (IEM) Genetic carriers
forensics
Restriction fragment length polymorphism (RFLP)
, what is it used for?
DNA fingerprinting
Used in forensic analysis, paternity testing and disease detection
Restriction fragment length polymorphism (RFLP), how does it work?
RFLPs are recognition sequences for restriction enzymes
By running DNA through a blot, similarities are observed
Can also be used to detect mutations
how often do genomes differ?
Genomes differ 1 in every 1000 base pairs
Variable number of tandem repeats (VNTR), what are they?
Pattern of short tandem repeats (STR) occurs in genome but varies in individuals
Useful in Identifying and severity of inherited disease
Huntington disease, fragile X syndrome, Frederick ataxia
Use of RLFP and VNTR in DNA typing ?
Prenatal diagnosis
Newborn screening
Carriers of disease
Production of recombinant proteins, what is it and how is it done?
Large scale production of recombinant proteins
cDNA of protein inserted into expression vectors and engineered to allow high levels of replication, transcription and translation to occur in a host
Large scale production and purification
what are recombinant proteins used to make?
Insulin Growth hormone Erythropoietin Clotting factors Vaccines
how has insulin been improved?
Normal human insulin has proline @position 28 and Lysine @position 29 on C terminus of B chain
Insulin Aspart (Novo Nordisk) -Proline 28 replaced by Asp
Both faster acting and more readily absorbed
Mix with normal insulin provides longer range of glycemic control
why are antibodies produced?
- Used as pharmacological drugs
- Used for research purposes
Monoclonal antibody (ab)- specific for a single epitope on antigen
Single clones of B lymphocytes fused with a tumor cell to make it immortalized
Humanization minimizes immunogenicity to prolong half life in patient
steps in antibody production?
- Immunization of mouse with immunogen peptide/protein
- Antibody secreting cells harvested from spleen (B cells, plasma cells)
- Fusion using eg PEG detergent
- Tissue culture -> immortalized tumor cells + abs= hybridoma secreting antibody
- Hybridomas screened for antibody production
- Ab producing hybridoma cloned
- Most efficient hybridoma clones producing most effective abs are chosen, harvested, and stored in liquid nitrogen and culture for production of abs when required
What does Elisa do?
Technique that tests for levels of specific antigen or antibody concentrations
Uses a corresponding antibody or antigen
Probe patient serum
Antigen or antibody is conjugated to a reactive enzyme (color-changing)
Antigen/antibody present -> color change observed
what is indirect elisa?
measures antibodies
what is sandwich elisa?
measures antigen
Measurement of cardiac-specific troponin isoforms, why is this relevant to elisa?
Troponin is the antigen in MI detection
Use sandwich ELIZA
Cardiac forms of T and I increase in MI
.Diagnosis of HIV infection, how is it done?
Specific antibody to HIV are produced
Indirect ELIZA is used
Can produce false-positives and false-negatives
Need confirmation with western blot
Hormone immunoassays, what do they do?
Measures levels of HCG
HCG is the antigen
Free HCG antibodies bind to HCG in urine complex moves to test site
Test site: immobilized HCG ab
Sandwich ELIZA
how is westernblot done?
- detects level of target protein
- SDS-PAge separates out proteins on a gel by using electric field
- transfer proteins to nitrocellulose membrane
- add primary antibody
- add secondary antibody-add substrate
- color shows
Early detection/confirmation of HIV infection, how?
Confirmation of HIV
Circulating levels of HIV p2 surface antigen appear even before antibodies are formed
Pregnacy test:, how is it done?
elisa
