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
measures amount of protein or antibody
Eastern Blotting
Detects post-translational modification on proteins
PCR
Subjected to high temp. to denature or separate single stranded DNA
Primers designed to complement sequences that flank each end of DNA in 3’-5’ direction
Allowed to anneal
Taq polymerase synthesizes copy of DNA by extending the primers on both ends
Advantage: Very small amount of template DNA needed
Disadvantage: Need to know the sequence of the flanking DNA for primer design, error prone
qPCR
- used to quantify copy number of specific copy number of a specific gene in two or more samples in real time
- this technique includes a probe which fluoresces only in presence of PCR product
- used to detect levels of infectious agent
Restriction fragment length polymorphism (RFLP)
o Individual genomes differ by 1 in every 1000 base pairs
o Some of these occur in the recognition sequences for restriction enzymes
o DNA fingerprinting
o Used in forensic analysis, paternity testing and disease detection
Ex: detection of sickle cell disease and number of restriction sites
b. Variable number of tandem repeats (VNTR)
o Varies in individuals
o Regions isolated from genomic samples by flanking restriction sites or through PCR
o Useful in identification and severity of inherited diseases
o Huntington disease, Fragile X syndrome, Frederich Ataxia detection
i. Improving on insulin
o Normal human insulin has proline at position 28 and lysine at position 29 at C terminus of B chain
o Lispro reverse position of these aa (made it faster)
o Insulin aspart- proline 28 replaced by aspartic acid
o Mixed with normal insulin, provides longer range of glycemic control
Abciximab
• Inhibits platelet aggregation
Baciliximab
• Prevents rejection of transplanted kidney
Cetuximab
• Treats metastatic colorectal cancer
Infliximab
• Treats autoimmune diseases
Retuximab
• Treats lymphomas, leukemias
Diagnosis of HIV
• Specific antibodies to HIV are produced in humans 4-6 weeks after infection
• Immobilized antigens in the wells with the biological samples
• Wash plate to remove unbound antibody and nonspecific antigen-antibody associations
• Add enzyme-linked antihuman IgG second antibody, wash, and add chromogenic substrates
• Development of color indicates presence of anti-HIV antibodies
- after sandwhich ELISA, then western blots to confirm