Chapter 9Molecular Diagnostics

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Chapter 9-Molecular Diagnostics

• Enzyme-Linked Immunosorbent Assay (ELISA)
• Monoclonal Antibodies
• DNA Diagnostic Systems (DNA fingerprinting)
• Molecular Diagnosis of Genetic Disease

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Old vs. New Molecular Diagnostics

• Old grow cells/pathogen-gttest
• 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 is also nice)

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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
bound primary antibody
antigenic site
Y
Y
colorless substrate
Y
Y
Target molecule
colored product
i i i i i i i i i i i i i i i
Support
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Fig. 9.2 Target antigens and polyclonal versus
monoclonal antibodies
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3
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Target antigen with various antigenic determinants
(epitopes)
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1
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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.
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Fig. 9.4 Procedure for producing a monoclonal
antibody to protein XNote 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 http//bcs.whfreeman.com/lodish5e/pa
ges/bcs-main.asp?vcategorys00010n06000i0601
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00900010000110001200013000140001500016000
1700018000190002000021000220002300099000n
s433
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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.)
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Fig. 9.5 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)

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Fig. 9.6 DNA diagnostic systems

• 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
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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.

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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 phosphase for
  detection (see Fig. 9.7)
• Note that fluorescent dyes can also be attached
  to DNA primers for detecting amplified DNA
  products (see Fig. 9.8)

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Fig. 9.9 Nonradioactive Hybridization Procedures
Molecular Beacons
Target DNA
(No Fluorescence)
Hybridization
Quencher
Fluorophore
Fluorescence!!!
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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

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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

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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?

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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 Lewinskys blue dress

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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

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Sequences examined in DNA fingerprinting

• VNTRs-variable number tandem repeats composed of
  8-80 bp repeat units (e.g., GCGCAATGn) 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., ACn) 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

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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

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Some examples of DNA fingerprinting

• Paternity cases
• Crime scenes

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Determining the probability of a match

• Relies on statistics
• Analysis depends upon your ethic background (i.e.
  African American, Caucasian, Hispanic Asian, etc.)

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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

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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?

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Fig. 9.14 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

Region of amplified DNA
Chromosomal DNA
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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
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Molecular Diagnosis of Genetic Disease

• Cystic fibrosis
• Sickle-cell anemia
• (see Fig. 9.17)