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TESTS TO DIAGNOSE DISEASE (DNA Microarrays (Idea: Can be used to test for…
TESTS TO DIAGNOSE DISEASE
DNA Microarrays
Idea: Can be used to test for genetic predisposition or to diagnose a disease from the hybridizing (or lack thereof) of complementary DNA. It can be used to test for the expression of many DNA sequences at once.
Definition of a DNA microarray (chip/biochip): a collection of short, known DNA sequences attached to a solid surface. Sample DNA from cells of a patient is washed across the surface. Binding at certain locations indicates the presence of pathogens.
Method: Isolate mRNA from patient cells. Then make DNA copies using reverse transcriptase (use primers and fluorescent nucleotides). Add the DNA to the microarray. Scan the microarray for fluorescence. The patient's DNA is often compared to a known sample DNA, using different colored tags, to see which sequences are common and which are unique between the two samples.
Example: "Normal" cell and tumor cells are hybridized using a microarray and scanned. Red spots are the patients DNA and green spots are the normal DNA. The frequency of red spots can show the severity of the cancer.
PCR
Idea: Can detect viral infections (influenza). It provides early diagnosis of viral infection. For people with compromised immune systems, early detection is vital.
Example: RNA virus (retrovirus, eg. HIV, influenza) enters cell and reverse transcribes its RNA into DNA and incorporates it into the host DNA. The virus contains glycoproteins which serve as antigens. The DNA that codes for these proteins can be amplified and detected using PCR.
Method: Heat (H-bonds break), Cool (primers are added), Warm (Taq polymerase is added). Cells from infected patient are sampled; RNA is extracted. PRIMER SEQUENCE that is specific to virus is added so that only the viral DNA will be amplified. Reverse transcription turns RNA into DNA. PCR amplifies DNA and is detected using electrophoresis.
Genetic Tests
Idea: Predisposition of genetic disease can be detected through the presence of
markers
Marker: Gene or DNA sequence with a known location on a chromosome that can be used to identify individuals with the potential to develop a disease. Can be observed in the analysis of Southern blots or gels.
Method: DNA from different samples cut with the same restriction enzyme and run on different lanes in gel. A mutated allele can be linked to repetitive sequences just upstream from the allele, which causes restriction enzymes to cut at different location, forming different length bands.
Examples: BRCA 1 and 2 found on chromosomes 17 and 13 can be mutated, resulting in breast cancer. Huntington's Disease results from dominant allele which can be identified using markers.
ELISA
Idea: Antigens and antibodies in blood are used to diagnose infection. Monoclonal antibodies are used to diagnose the infection. They bind to antigens of the pathogen or to antibodies that humans produce to fight against the pathogen.
Definition Enzyme linked Immunosorbent Assay. It is a technique used to detect proteins (e.g. antigens or antibodies) in the blood to determine if infection from a pathogen has occurred.
Method: To test for antigens, an antibody specific to the antigen is fixed to a surface. It is exposed to the patients blood. If the antigen is present, it binds to the antibody. Then, an antibody (labeled antibody) attached to an enzyme is added. Finally, substate is added and the color of the substrate changes if enzyme is present. This is all done in wells containing different patients' blood samples. The extent to which the well changes color shows the concentration of the antigen in the blood.
To test for antibodies, the same procedure is done, however antigens are fixed to the surface so that antibodies in the blood attach to them. Labeled antibodies are still used, and substrates are added.
Tracers
Idea: Tracers are used in tracking experiments that provide information about the localization and interaction of a desired protein. A PET scan is used to detect the fluorescent trackers.
Example: Transferrin linked to luminescent probes is used to track tumor cells.
Method: Transferrin injected into vein. It travels through the body. It accumulates in organs and tissues that use transferrin (particularly for the iron). The patient is scanned for positron emission from the fluorescent label attached to the transferrin. Tumor cells have high iron uptake, so transferrin is seen at higher quantities in tumor cells.
Note: Bioluminescence can be used to detect tumors, and they are harmless.
Example: Radioactive fluorine to detect tumors in lymph nodes.
Radioactive fluorine is attached to a larger molecule that is injected into body to target enzymes active in tumors. It indicates the activity of enzyme thymidine kinase.