Chapter 9-Molecular Diagnostics
•Enzyme-Linked Immunosorbent Assay (ELISA)
•Monoclonal Antibodies
•DNA Diagnostic Systems (DNA fingerprinting)
•Molecular Diagnosis of Genetic Disease
Old vs. New Molecular Diagnostics
• Old: grow cells/pathogen->test
• Such growth can be a problem as it is
sometimes slow, costly, and specific
• New: direct test (either immunological or DNA
based)
• Detection must be: specific, sensitive, and
simple (fast and automatable are also nice)
Fig. 9.1 Enzyme-Linked Immunosorbent Assay
(ELISA): immunological detection
A. Bind sample to the support (commonly plastic or a membrane)
B. Add primary antibody; wash
C. Add secondary antibody-enzyme conjugate; wash
D. Add substrate
enzyme linked
secondary antibody
iiiiiiiiiiiiiii
E
colorless substrate
Y
Y
Y
Y
Target molecule
antigenic site
E
Y
Y
Support
E
Y
Y
E
bound primary
antibody
colored product
Fig. 9.2 Target antigens and polyclonal versus
monoclonal antibodies
2
1
3
4
Target antigen
with various antigenic
determinants (epitopes)
7
5
6
Polyclonal antibodies are made against and react with
multiple antigenic sites (epitopes) on a target antigen.
Monoclonal antibodies are directed against a particular
antigenic site.
Fig. 9.4 Procedure for
producing a monoclonal
antibody to protein X
Note: B lymphocytes or B cells produce
antibodies but do not reproduce in
culture. Some B cells can become
cancerous and are known as myelomas
which can reproduce in culture.
See Chapter 9 animation
http://bcs.whfreeman.com/lodish7e/#
800911__811944__
Fig. 9.3 Explanation of how HAT medium works
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
• Polypeptide hormones (chorionic gonadotropin,
growth hormone)
• Tumor markers (Prostate-specific antigen)
• Cytokines (interleukins 1-8)
• Drug monitoring (cyclosporin)
• Miscellaneous targets (Vitamin B12)
• Infectious diseases (Chlamydia, Herpes, Rubella,
Hepatitis B, Legionella, HIV)
Fig. 9.6 DNA diagnostic systems
1.
2.
3.
4.
Bind ssDNA (target) to membrane
Hybridize to labeled ssDNA or RNA (probe)
Wash membrane to remove unbound probe
Detect hybrid sequences formed between the
probe and target DNA (concern: false +s & -s)
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
• Use of biotin-labeled nucleotides in DNA probes
instead of 32P, then add avidin (streptavidin) which
binds to biotin, and then add biotin attached to an
enzyme like alkaline phosphatase for detection (see
Fig. 9.11)
• Note that fluorescent dyes can also be attached to
DNA primers for detecting amplified DNA products
(see Fig. 9.12)
Nonradioactive Hybridization Procedures
Fig. 9.13 Nonradioactive Hybridization
Procedures: Molecular Beacons
Target DNA
Molecular beacon probe
(No Fluorescence)
.
Hybridization
Quencher
Fluorophore
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
• You're 99.9% identical
• But of course, you are unique--in a genome of three
billion letters, even a 0.1 % difference translates into
three million differences.
• These differences (or polymorphisms) reside in
several places in the genome, often in microsatellites
• Examples of such polymorphisms include VNTRs,
STRs, RFLPs and SNPs
DNA Fingerprinting
• Focuses on the 0.1-1.0% of human DNA that is
unique
• First described in 1985 by Dr. Alec Jeffreys in England
• DNA evidence is admissible in courts
• Labs such as Cellmark Diagnostics and Lifecodes
Corporation are examples of companies which
provide such DNA evidence to courts, but states and
many U.S. cities have labs for DNA fingerprinting
• Have any of you worked in a crime lab?
Uses of DNA fingerprinting
• Paternity testing
• Identification of criminals (e.g. murderers, rapists,
letter bombers)
• Immigration disputes (family relationships)
• Identification of deceased individuals with mutilated
or decomposed bodies (e.g., the military, 9/11 victims)
• Identifying the sperm donor who “decorated” Monica
Lewinsky’s blue dress
How is DNA fingerprinting done?
• DNA obtained from hair, semen, blood, sweat, saliva,
bone or any other tissue (often found at a crime scene)
• Can be done by southern blotting with an appropriate
probe or by a PCR method using appropriate primers
• Can use single locus probes/primers or multilocus
probes/primers
• DNA can be resolved on a gel or by a capillary
electrophoresis system
Sequences examined in DNA fingerprinting
• VNTRs-variable number tandem repeats; composed of 880 bp repeat units (e.g., [GCGCAATG]n) which are
tandemly repeated so that the overall length is 1-30 kb
• STRs-short tandem repeats; composed of 2-7 bp repeat
units (e.g., [AC]n) which are tandemly repeated so that
the overall length is less than 1 kb
• RFLPs-restriction fragment length polymorphisms
• SNPs-single nucleotide polymorphisms
• Mitochondrial DNA-maternal inheritance, tends to be
more stable than nuclear DNA
• Y chromosome DNA- passed from father to son
DNA fingerprinting: an example
• D1S80, a VNTR located on human chromosome 1,
contains a 16 bp repeat unit
• The number of repeats varies from one individual to the
next, and is known to range from 14-41
Some examples of 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)
• Use of arbitrary oligonucleotide primers,
usually 9-10 nucleotides long, in a PCR of total
DNA to distinguish plant cultivars, animal
varieties, and microbe isolates
• A PCR product will be produced whenever two
of the oligonucleotide primers face one
another and are 100-3,000 bp apart
Chromosomal DNA
Region of amplified DNA
Fig. 9.20 Real Time PCR
• 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
• One example involves using Pseudomonas
fluorescens (genetically engineered for
bioluminescence) to monitor pollutants
• If pollutants are present in a sample, then cell
death occurs and “the light goes out”
lux genes in the
chromosomal DNA
Fig. 9.5 Bacterial biosensors (another example)
• Green fluorescent protein (GFP) can be used a
reporter gene under the control of some inducible
promoter (e.g., one that responds to some
environmental signal such as a toxin)
• If the signal is present GFP will be produced
Molecular Diagnosis of Genetic Disease
• Cystic fibrosis
• Sickle-cell anemia
• (see Fig. 9.28)