Molecular diagnostics Flashcards
Techniques to Detect Infectious Agents and Diagnose Inherited Disorders
- Hybridization
- Polymerase Chain reaction (PCR)
hybridization steps
-Single stranded DNA binds to another strand of DNA or RNA with
complementary sequence to form DNA-DNA hybrid or DNA-RNA
hybrid.
-detection and quantification of target DNA or RNA
-single-stranded oligonucleotides (called probes) are
designed and synthesized.
- target DNA converted to SS and immobolized
- hybridized to complementary sequences in samples
southern blotting
- both probe and nucleic acid are DNA
-Determine which
restriction fragments are
associated with a gene
northern blotting
probe is ss DNA and target is mRNA -Measure size and quantities of mRNA molecules (questions about gene expression
western blotting
-target is protein
-Measures amount of
protein or antibody
polymerase chain reaction (PCR) steps
-Double stranded DNA (ds DNA) obtained from a
patient/individual/pathogen
-denature
-primers flank end of DNA
-primers anneal
-add all 4 dNTPS
-taq polymerase : synthesizes copy of DNA
PCR advantage
-Very small amount of template DNA needed, 109-fold
amplification from trace amount of DNA
PCR disadvantage
- need to know sequence of flanking DNA for primer design
- error prone
- amplification of contaminating DNA
quantitative PCR (qPCR) overview
-Used to quantify copy number of a specific gene in two or more samples in real time. - In addition to primers, this technique includes a probe which fluoresces only in presence of the PCR product -Probe usually a complementary
what is qPCR used for?
- Detect levels of an infectious agent
- Determine levels of gene expression
what can you use to detect variations in DNA sequences
1) Restriction Fragment Length Polymorphism (RFLP)
2) Variable number of tandem repeats (VNTR)
- forensic and diagnostic
Restriction Fragment Length
Polymorphism (RFLP)
-diff in genomes based on recognition for restriction enzyme
-DNA fingerprinting
-Used in forensic analysis,
paternity testing and disease
detection
what can you use to detect mutations in offspring passed on by parent?
RFLP
Variable number of tandem repeats (VNTR)
-Pattern of short tandem repeats (STR) occurs in
genome but varies in individuals
-Useful in identification and severity of inherited
diseases
-Huntington disease, Fragile X syndrome,
Frederich Ataxia
How could you diagnose Huntington’s disease?
using VNTR
what are recombinant proteins used for?
medications such as: Insulin growth hormone erythropoietin clotting factors vaccines against diseases such as flu and malaria, viral infections
how are recombinant proteins produced?
- take plasmid of gene of interest and insert cDNA into expression vector
- bacteria internalize plasmid with gene of interest
- grow bacteria to make more plasmic
- gene translated to protein
what are two modifications that improve the action of insulin
-lispro: reversed postion of 2 a.a on normal human insulin
-Insulin aspart (Novo Nordisk) – proline 28 replaced by aspartic acid.
-Both Lispro and Insulin aspart faster acting, more readily absorbed.
-Mix with normal insulin, provides
longer range of glycemic control
monoclonal Antibodies
specific for a
single epitope on antigen
what is the production of antibodies in lab used for?
drugs (cancer,
rheumatoid arthritis, Crohn’s
disease, hepatitis)
Enzyme-linked Immunosorbent Assay
ELISA
-tests for levels of specific antigen or antibody concentrations
Indirect ELISA
measures amount of antibody in sample
sandwich ELISA
measures amount of antigen in sample
what technique could you use to diagnose HIV
indirect ELISA
-looks at amount of antibody formed
what technique could you use to diagnose MI
sandwich ELISA
-mesures antigen (cardiac forms of T and I increase in acute myocardial infarction
what technique do pregnancy tests use?
sandwich ELISA
- looks for hCG which is an antigen
- antibody built in stick
what is Western Blotting (immunoblotting) used for?
-to detect levels of target protein
what are steps to western blotting
- SDS page to separate proteins
- transfer proteins from gel to nitrocellulose
- add primary antibody
- add secondary antibody
- secondary antibody= enzyme tag which gives color
after diagnosing HIV how could you CONFIRM it
western blot
