Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Y
Target molecule
Y
E
E
Support
Fig. 9.2 Target antigens and polyclonal versus
monoclonal antibodies
2 3 4
Target antigen 5
with various antigenic
1 determinants (epitopes)
7 6
Myeloma cells are HGPRT- and will die on HAT media having hypoxanthine,
aminopterin (an antifolate), and thymidine.
Spleen cells are HGPRT+ , so spleen-myeloma (hybridoma) cells can grow on HAT.
(Note: spleen cells by themselves cannot grow in culture.)
Table 9.2 Targets for diagnostic monoclonal antibodies
membrane
DNA based diagnosis of Malaria and
Typanosoma cruzi
• A DNA probe from a highly repeated DNA sequence
of Plasmodium falciparum, the parasite that causes
malaria, is used to screen blood samples via
hybridization assays
• DNA primers are made against the ends of a 188 bp
repeated sequence contained in the protozoan
parasite Typanosoma cruzi, the causative agent of
Chagas disease and used in a PCR/polyacrylamide gel
electrophoresis detection method
• Other examples of DNA-based detection: Salmonella
typhi (food poisoning), certain E. coli (gastroenteritis),
Mycobacterium tuberculosis (tuberculosis), etc.
Nonradioactive Hybridization Procedures
Hybridization
Fluorophore Quencher
Fluorescence!!!
DNA Fingerprinting & Forensics
• History
• Uses of DNA Profiling
• Hypervariable DNA sequences examined (RFLPs, VNTRs,
STRs, SNPs, mitochondrial DNA, Y chromosomal DNA)
• Methods (Southerns & PCR)
• Statistical considerations
• Technical considerations
• Databases and Privacy
DNA Fingerprinting
• Paternity cases
• Crime scenes
Determining the probability of a match
• Relies on statistics
• Analysis depends upon your ethic background
(i.e. African American, Caucasian, Hispanic
Asian, etc.)
Technical Considerations
• Preserve the integrity of DNA sample
• Avoid DNA contamination & degradation
• Avoid incomplete digestions if REs are used
• Use standard hybridization conditions
• Use standard PCR primers and procedures
• Gel analysis is less reproducible than capillary
electrophoresis of PCR products
• Difficulties in interpreting bands on a gel or X-ray film
DNA databases
• Already in place in the FBI for convicted felons (i.e.,
CODIS-COmbined DNA Index System, involves 13 STR
loci) and the Dept. of Defense for armed service
personnel and the Virginia saliva and blood bank of
convicted felons
• A national DNA database has been suggested. What
do you think?
• Could current or potential employers or insurance
companies base decisions they make on this kind of
data?
Fig. 9.18 Random Amplified Polymorphic DNA (RAPD)
• A way to quantitate
DNA in a PCR
• Involves the use of
SYBR green dye
• SYBR green only
binds to and
fluoresces with
dsDNA
Fig. 9.16 Bacterial biosensors
• Cystic fibrosis
• Sickle-cell anemia
• (see Fig. 9.28)