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

Target molecule

antigenic site

i i i i i i i i i i i i i i i

Support

A. Bind sample to the support (commonly plastic or a membrane)B. Add primary antibody; wash

C. Add secondary antibody-enzyme conjugate; washD. Add substrate

Y

YY

Y

bound primaryantibody

Y

E Y EYEYE

enzyme linkedsecondary antibody

colorless substrate

colored product

Fig. 9.2 Target antigens and polyclonal versus monoclonal antibodies

Polyclonal antibodies are made against and react withmultiple antigenic sites (epitopes) on a target antigen.Monoclonal antibodies are directed against a particularantigenic site.

Target antigenwith various antigenicdeterminants (epitopes)1

2 3 4

5

67

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.

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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.)

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)

Fig. 9.6 DNA diagnostic systems

1. Bind ssDNA (target) to membrane2. Hybridize to labeled ssDNA or RNA (probe)3. Wash membrane to remove unbound probe4. 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. 9.9 Nonradioactive Hybridization Procedures: Molecular Beacons

Target DNA

.

Molecular beacon probe

Hybridization

Fluorophore Quencher

Fluorescence!!!

(No 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 8-80 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 thechromosomal 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)