Lec 4 molecular diagnsostics Flashcards
What are some techniques to detect infectious agents and diagnose inherited disorders?
Hybridization
polymerase chain reaction (PCR)
What must be known for hybridization and PCR?
know the sequence of the pathogen or gene
(5000 pathogens sequenced human genome sequenced)
What is hybridization
single stranded DNA binds to DNA or RNA with complementary sequence DNA-DNA hybrid
or DNA-RNA hybrid
how does hybridization happen?
target DNA converted to single stranded DNA and imobilized (blotting)
single-stranded oligonucleotides (probes)
added and attach to complimentary region
Southen Blotting
probe and target nucleic acid are DNA
determine which restriction frags are associated with a gene
Northern blotting
probe is single strand DNA and target is mRNA
measure size and quantities of mRNA molecules
western blotting
target is a protein
measure amount of protein
Eastern blotting
PTM (post translational modifications is target
PCR
polymerase chain reaction
- double-stranded DNA from target
- high temp denature/separate
- primers complement sequences in flank each end of DNA (3’-5’)
- anneal (bind DNA)
- add deoxy nucleotide triphosphates (dNTPS) all 4
- Taq polymerase synthesizes copy of DNA from primer
- thermocycler (does temp, number of cycles)
advantages and disadvantages of PCR
A-small amount of template DNA needed to amplify
D-need to know sequence of flanking DNA for primer, error prone, amp of contaminating DNA
qPCR
quantitative PCR
quantify copy number
primers added and a probe which flouresces in presence of PCR product
(complementary oligo with tag)
Detect levels of infectious agent/ levels gene express
Techniques used to detect variation in DNA sequences
Restriction Fragment Length Polymorphism (RFLP)
Variable number of tandem repeats (VNTR)
RFLP
restriction fragment length polymorphism
genomes differ 1 in 1000 base pairs
some in recognition sequences for restriction enzyme
cleaved with restriction enzyme show two differnt pattern for different genomes
DNA fingerprinting
What is RFLP used in?
forensic analysis
paternity testing
disease detection
VNTR
variable number of tandem repeats
short tandem repeats in genome but varies by individual
VNTR repeat regions isolated by flanking restriction sites though PCR
What is VNTR used in?
identification and severity of inherited diseases
Huntington disease!! fragile x syndrome frederich ataxia
recombinant proteins examples
- insulin
- growth hormone
- erythropoietin
- clotting factors
- vaccines (flu/viral)
What makes a recombinant protein
cDNA of protein inserted into expression vectors
for ex. plasmid vector from bacteria which is then replicated by bacteria making proteins and end products
monoclonal antibody targets and uses
- inhibit platelet agg
- prevents rejection transp kidney
- treat metastatic colorectal cancer
- autoimmune diseases treat
- treat lymphomas, leukemias
- (many uses)
ELISA
enzyme-linked immunosorbent assay
tests for levels of specific antigen or antibody concentrations using anitbody or antigen
Indirect ELISA
Sandwich ELISA
ELISA applications
- Diagnose HIV
- MI detection
- pregnancy test
ELISA and HIV
(indirect ELISA)
antibodies to HIV produced 4-6 weeks
efficient, sensitive, cost efficient
NEED CONF with western blotting
ELISA and MI detection
cardiac form of troponin increase (antigen)
(sandwich ELISA)
which binds to the antibody
ELISA and pregnancy test
Sandwich ELISA
hCG antibodies (free)
bind hCG in urine
complex moves to test site that has immoblized hCG antibody
sandwich complete dye gives color
control site dye gives color regardless (non-specific antibody)
confirm test working
Western Blotting
immunoblotting
- transfer protein to SDS page
- add primary antibody (binds target)
- wash excess
- add secondary antibody (binds primary)(enzyme tag)
- wash excess
- use visualization to see enzyme tag
application of western blot?
confirmation of HIV
circulating HIV p24 surface antigen (proteins)
and other antigens (proteins)
can use controls to compare samples detect antigens
