Molecular Diagnostics Flashcards
southern blotting
both probe and target nucleic acid are DNA
northern blotting
probe is single stranded DNA and target is mRNA
western blotting
target is protein or antibody
how does PCR work
dsDNA obtained from patient/individual/pathogen is denatured with heat and separated to ssDNA
primers flank each end of DNA and are allowed to anneal in cooler temps
deoxynucleotide triphosphates (dNTPS) are added
Taq polymerase synthesizes copy of DNA
Taq polymerase
synthesizes copy of DNA by extending the primers on both ends; DNA doubles in each cycle and becomes greatly amplified
advantages of PCR
very small amount of DNA is needed for the template; 10^9 fold increase from original DNA
disadvantages of PCR
you need to know the sequence of the flanking DNA for primer design
it’s error prone
you could amplify contaminated DNA
quantitative PCR (qPCR)
used to quantify copy number of a specific gene in two or more samples in real time
in addition to primers, it uses a probe which fluoresces only in presence of the PCR product
used for: detecting levels of an infectious agent and determining levels of gene expression
restriction fragment length polymorphism (RFLP) steps (5)
DNA fingerprinting; results is bands of DNA on nylon membrane
- DNA is cleaved by restriction endonucleases
- resolve DNA fragments on agarose gel
- transfer DNA to nylon membrane
- add radioactive DNA probe; wash off excess probe
- expose x-ray film to nylon membrane
how is sickle cell anemia shown on an RFLP
patients with sickle cell only have two restriction sites in their DNA, so their RFLP will only show one fragment on the membrane
normal beta globulin allele has 3 Ddel restriction sites and will show two fragments
variable number of tandem repeats (VNTR)
short tandem repeats (STR) occurs in normal genome and varies in individuals
VNTR repeat regions are isolated from genomic samples by flanking restriction sites or through PCR
useful in identification and severity of inherited diseases
ex: huntington disease, fragile X syndrome, frederich ataxia
how huntington’s is diagnosed
uses VNTR
PCR is done on healthy individual and patient with huntington’s; alpha-32 probe is attached to dCTP
resolve DNA fragments on urea-polyacrylamide gel
expose x-ray film to dried gel
huntingon’s will show much higher CAG segments
recombinant proteins
engineered to allow high levels of replication, transcription, and translation to occur in a host; cDNA of the protein is inserted into expression vectors
large scale production and purification
used to make insulin, growth hormones, erythropoietin, clotting factors, vaccines
how human insulin production works
insulin producing gene is isolated from pancreas cells
plasmid DNA is extracted from a bacterium and cut with restriction enzymes, forming plasmid vector
human insulin producing gene is inserted into the bacterial plasmid vector to form the recombinant DNA
how does Lispro improve insulin use
reverses the position of proline at position 28 with lysine at position 29 at C terminus of B chain
works in 2-4 hours instead of 6-8 hours
how does insulin aspart improve insulin use
the proline at 28 is replaced with aspartic acid
works in 2-4 hours instead of 6-8 hours
monoclonal antibody
specific for a single epitope on antigen
used to treat many cancers
how antibodies are generated
mouse is immunized with immunogen peptide protein
antibody secreting cells are harvested from the spleen
mouse spleen cells are fused with myeloma cells to form hybridomas
the cells are grown in culture in HAT medium
cells divide infinitely because they are cancer cells, which causes infinite production of the monoclonal antibody
abciximab
chimeric monoclonal antibody
inhibits platelet aggregation
baciliximab
chimeric monoclonal antibody
prevents rejections of transplanted kidney
cetuximab
chimeric monoclonal antibody
treats metastatic and colorectal cancers
infliximab
chimeric monoclonal antibody
treats autoimmune disease
retuximab
chimeric monoclonal antibody
treats lymphomas, leukemias
ELISA
immunological tests which tests for the levels of specific antigen or antibody concentrations in biological samples using a corresponding antibody or antigen; quantitative
uses fluorescence to determine quantitative results
indirect ELISA
measures the amount of an antibody in a sample
sandwich ELISA
measures the amount of an antigen in a sample
how ELISA works
microplate is pre-coated with antibody
target protein will be captured (bound) to the antibody when added to the well
second antibody conjugated with horseradish peroxidase (HRP) is added and binds to captured protein
unbound second antibody is washed away and the HRP substrate TMB is added which develops blue color
how ELISA diagnoses HIV
specific antibodies to HIV are produced 4-6 weeks after infection
uses indirect ELISA to search for the antibodies in the body
how ELISA can detect MI
troponin is the antigen in this case
uses sandwich ELISA to find the antigen
how ELISA works for a pregnancy test
looking for HCG (the antigen)
uses sandwich ELISA to look for HCG
the test stick has immobilized hCG antibody complex, which will bind to hCG in urine to complete the sandwich which produces the colored line
western blotting (immunoblotting)
used to detect the levels of a target protein in a biological sample
mechanism of western blotting
protein samples are placed in SDS polyacrylamide gel
proteins are transferred to nitrocellulose membrane where they are exposed
primary antibody is added which binds to the target protein
tagged secondary antibody is added which binds to primary antibody
visualization agents/chemicals are used to detect secondary antibody signal
what antigen of HIV does western blotting detect
detects HIV p24 surface antigen
other antigens include:
- gp160
- gp120
- p66
- p66
