Molecular diagnostics

CHC 812: CELL BIOLOGY, GENETICS, IMMUNOLOGY AND TERATOLOGY

Lecture 01, 2015Molecular Diagnostics

Lecturer: Dr. G. Kattam Maiyoh

GKM/M.MEDPAEDS/LECT 01/2015

Molecular Diagnostics

The use of molecular biology techniques to

expand scientific knowledge of the natural

history of diseases, identify people who are at

risk for acquiring specific diseases, and

diagnose human diseases at the molecular

level.


Molecular Diagnostic

• USE OF:– Sequence Specific INFORMATION • in

– MACROMOLECULES• for

– Risk identification– Diagnosis– Prognosis– Prediction of response to therapy– Monitoring therapeutic responses


Macromolecules

• Peptides/proteins

• Polysaccharides

• Polynucleotides /nucleic acids


Molecular Diagnostics: Significance

To face the near future, the medical practitioner not only understand molecular biology, but must also embrace the use of this rapidly expanding body of information in his medical practice, whether practicing family medicine pediatrics, oncology, obstetrics and gynecology, pathology, or any other medical specialty.


Molecular Diagnostics are Transforming Medicine

Pre-natal testing

Disease predisposition

Disease detection

Drug selection

Recurrence monitoring

Key questions

-> Need for Molecular tests

“Is the baby healthy? “

“What diseases is this patient at risk for?”

“Does this patient have a disease?”

“What drugs should I prescribe?”

“Has the disease returned?”

Molecular diagnostics is >$3 billion

market WW and growing at >20%

annually


Old vs. New Molecular Diagnostics

• Old: grow cells/pathogen->test• Such growth can be a problem as it is

sometimes slow AND costly.• New: direct test (either immunological or

DNA/RNA based)


Characteristics of a Detection System

• A good detection system should have 3 qualities:♣ Sensitivity♣ Specificity♣ Simplicity

• Sensitivity means that the test must be able to detect very small amounts of target even in the presence of other molecules.

• Specificity: the test yields a positive result for the target molecule only.

• Simplicity: the test must be able to run efficiently and inexpensively on a routine basis.

Molecular Diagnostics


Molecular DiagnosticsImmunological Diagnostics Methods

1. Radioimmunoassay

2. Enzyme-Linked ImmunoSorbent Assay (ELISA)

3. Western Blotting

4. Immunoprecipitation

5. Immunofluorescence

6. Flow Cytometry and Fluorescence

7. Alternatives to Antigen-Antibody Reactions

8. Immunoelectron Microscopy


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


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)


Radioimmunoassay



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; washC. Add secondary antibody-enzyme conjugate; wash

D. Add substrate

Y

YY

Y

bound primaryantibody

Y

E Y EYEYE

enzyme linkedsecondary antibody

colorless substrate

colored product


Immunological Diagnostics Methods - ELISA

• Addition of a specific antibody (primary antibody) which will bind to the test molecule if it is present.

• Washing to remove unbound molecules.• Addition of secondary antibody which will

bind to the primary antibody.• The secondary antibody usually has attached

to it an enzyme e.g. alkaline phosphatase.• Wash to remove unbound antibody.• Addition of a colourless substrate which will

react with the secondary antibody to give a colour reaction which indicates a positive result.

-> can be used for quasi High-throughput!!!


ELISA -Variants

Detection based on enzyme catalyzed reactions:

1.alkaline Phosphatase2.horseradish peroxidase3. β-galactosidase

Detection based on fluorescent labeled secondary antibody


ELISA –VariantsThe ELISPOT

The ELISPOT assay -> to determine

quantitatively the # of cells in a population

that are producing specific Ab or cytokine.

-> precipitates & forms a spot only on the areas of the well where cytokine-secreting cells had been deposited.

P.T.O



Western blot

SDS-Page: separates the components according to their molecular weight.

Blot: the proteins in the gel are transferred to the sheet of nitrocellulose or nylon by the passage of an electric current.

Immunoreaction: probed with Ab & then radiolabeled or enzyme-linked 2nd Ab.

Detection: a position is visualized by means of an ELISA reaction.




ImmunoprecipitationImmuno-precipitates can be collected using magnetic beads coupled to a secondary antibody.


Immunofluorescence

Fluorochromes-Fluorescein (490 517nm)→-Rhodamine (515 546nm)→-Phycoerythrin

mIgM-producing B cells indirectly stained with rhodamine-conjurated secondary Ab under a fluorescence microscope.

Protein A has the ability to bind to IgG


Immuno Electron Microscopy

electron-dense labelsabsorb electrons.

An immunoelectronmicrograph

of the surface of a B-cell

lymphoma was stained with two

antibodies (Ab against class II

MHC labeled with 30nm gold

particles, & another Ab against

class I MHC w/ 15nm gold

particles.

(The density of class I exceeds

that of class II)

- Electron-dense label (ferritin

or colloidal gold) is conjugated

to the Fc

portion.GKM/M.MEDPAEDS/LECT 01/2015

We now know how God wrote the book of life

Bill Clinton

But do we know how to read the book ?

Molecular Genetic Tests• Genetic test:– Analyis of human• DNA• RNA• chromosomes• proteins• metabolites

– to detect heritable disease-related• genotype, • phenotype• karyotype

– for clinical purposes.


Genetic Diagnosis“Purpose”

• Diagnostic Testing• Screening • Presymptomatic Testing• Prenatal testing• Preimplantation Diagnosis• Pharmacogenetic testing • Susceptibility to environmental agents


Genetic Alterations

• Chromosomal alterations• “Gene-level” alterations.


Preimplantation Diagnosis/ Screening

• Prenatal diagnosis or prenatal screening (note that prenatal diagnosis and prenatal screening refer to two different types of tests) is testing for diseases or conditions in a fetus or embryo before it is born.



Preimplantation Diagnosis

DNA diagnostic systems

1. Bind ssDNA (target) to membrane

2. Hybridize to labeled ssDNA or RNA (probe)

3. Wash membrane to remove unbound probe

4. Detect hybrid sequences formed between the probe and target DNA (concern: false +s & -s)

membrane


Ordering Molecular Tests• Patient preparation: None

– Avoid heparin: interferes with PCR.

• Specimens:– Fresh whole blood: EDTA/Citrate– Fresh tissues– Frozen tissues– Paraffin embeded tissues– Slides etc.


Ordering Molecular genetic Tests

• Specimen Handling• DNA-based tests:

– Room temperature, up to 72 hours (maybe more with special buffers)

• RNA-based tests:– Deliver ASAP (4-6 hours)– Special considerations for proprietary test.


Ordering Molecular genetic Tests • Essential info:

– Clinical information – pedigree, if possible– Race– reason for testing.

• Informed consent:• Nature of test; availability of genetic counseling;

implications of positive and negative tests, etc.


Molecular Diagnosis of Genetic Disease

• Cystic fibrosis Sickle-cell anemia


DNA based diagnosis of Malaria and Typanosoma cruzi

1. 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

2. 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. GKM/M.MEDPAEDS/LECT 01/2015

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


In case of lack of Hybridization probesAre washed away henceNo signal

Fig. 9.13 Nonradioactive Hybridization Procedures: Molecular Beacons

Target DNA

.

Molecular beacon probe

HybridizationFluorophore 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

– Variable number tandem repeats– Short Tandem Repeats– Restriction fragment length polymorphism– Single Nucleotide Polymorphism


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


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, bomb blast)• Identifying the sperm donor who “decorated” Monica

Lewinsky’s blue dress


Preparation of a DNA fingerprintStep 1

• Specimen collection– blood, semen, etc– Easy to contaminate a DNA sample with DNA from

other sources (bacteria, DNA of person collecting sample)

– DNA is not stable for very long-it degrades• sunlight• heat• moisture

January 23, 2015 47GKM/Forensic and Clinical Bioc./Lec

03/2013

• DNA fingerprinting is a comparative process:– DNA from crime scene is compared with DNA of a

suspect– So minimum of two samples must be prepared

Step 2• DNA extraction

– standardized methods have been developed– need to separate DNA from other cell material

and debris from crime scene.


03/2013

Step 3

• PCR using primers targeting STRs at different loci• PCR amplify STRs using target sites on

chromosome


03/2013

Step 3 PCR amplification of DNA

1 strandof DNA

Heat todenaturedouble-strandedDNA

Design primers that anneal to STR locus

Amplify all the regions of the chromosomewhere the STRs exist.

STR locus

STR locus


03/2013

PCR allows you to make millions of copies of the STR region from a single copy of DNA you recovered from crime scene.


03/2013

• Since the # of times sequence is repeated is different for each person, fragment size will be different.

• This is done for 13 different STR sequences• Differences occur among individuals at each of

the 13 loci on the chromosome where the STRs occur

• This allows for a lot of variation


03/2013

Restriction Fragment Length Polymorphism

G-G-C-C-X-X-X-G-G-C-C-X-X.. G-G-G-C-C-X-X-G-G-C-C-X-X…..

STR

C-C-X-X-X-G-G C-C-X-X-G-G

PCR amplifySTR region

STR

well well

Gelelectrophoresis

Person A Forensic sample

For 1 STR sequence at 1 locus


03/2013

• If you do this for 13 different repeat sequences at 13 different loci on the chromosome, each person produces a different band pattern when the fragments are separated by gel electrophoresis

• Banding patterns are identified using specific probes (see next slide)

• Since the patterns are unique to an individual, they are referred to as DNA finger prints


03/2013

Banding Patterns

Some examples of DNA fingerprinting

• Paternity cases• Crime scenes


Example

January 23, 2015GKM/Forensic and Clinical Bioc./Lec

03/201356

E – reference sample, S1 – suspect 1 and S2 – suspect 2



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


Test Choice

• Cost• Sample requirements• Turnaround time• Sensitivity/Specificity• Positive/ Negative predictive value• Type of mutation detected• Genotyping vs mutation scanning


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?


• A way to quantitate DNA in a PCR

• Involves the use of SYBR green dye

• SYBR green only binds to and fluoresces with dsDNA (detect product)


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


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


THE END


Health & Medicine

Molecular diagnostics