Molecular Diagnostics

The use of molecular biology techniques to

1) expand scientific knowledge of the natural
history of diseases,
2) identify people who are at risk for
acquiring specific diseases, and
3) diagnose human diseases at the molecular
level.
Molecular Diagnostic
• USEOF:
– Sequence Specific INFORMATION
• in
– MACROMOLECULES
• for
– Risk identification
– Diagnosis
– Prognosis
– Prediction of response to therapy
– Monitoring therapeutic responses

GKM/M.MEDPAEDS/LECT 01/2015
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
-> Need for Molecular tests
Molecular
diagnostics is
>$3 billion Recurrence monitoring
market WW and
growing at >20%
annually
Drug selection

Disease detection

Disease predisposition

Pre-natal testing Key questions

“Is the baby “What diseases “Does this “What drugs “Has the disease
healthy? “ is this patient at patient have a should I returned?”
risk for?” disease?” prescribe?”
Old vs. New Molecular Diagnostics
• Old: grow cells/pathogen->test
• Such growth can be a problem as it is
sometimes slow ANDcostly.
• New: direct test (either immunologicalor
DNA/RNAbased)
Molecular Diagnostics
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
Immunological Diagnostics Methods

1. Radioimmunoassay
2. Enzyme-Linked ImmunoSorbent Assay (ELISA)
3. Western Blotting
4. Immunoprecipitation
5. Immunofluorescence
6. Flow Cytometry and Fluorescence
7. Immunoelectron Microscopy
Target antigens and polyclonal versus
monoclonal antibodies

2 3 4

Target antigen 5
withvarious antigenic
1 determinants(epitopes)

7 6

Polyclonal antibodies are made against and react

wit
h multiple antigenic sites (epitopes) on a target
antigen.
Monoclonal antibodies are directed against a particular
Targets for diagnostic monoclonal antibodies
• Polypeptide hormones (chorionic gonadotropin,
growth hormone)
• Tumor markers (Prostate-specific antigen)
• Cytokines (interleukins 1-8)
• Miscellaneous targets (Vitamin B12)
• Infectious diseases (Chlamydia, Herpes, Rubella,
Hepatitis B, Legionella, HIV)
 Molecular Genetic Tests
• Genetic test:
– Analyis of human
• DNA
• RNA
• chromosomes
• proteins
• metabolites
– to detect heritabledisease-related
• genotype,
• phenotype
• karyotype
– for clinical purposes.
 Genetic Diagnosis
“Purpose”

• Diagnostic Testing
• Screening
• 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 isborn.
Preimplantation Diagnosis

GKM/M.MEDPAEDS/LECT 01/2015
DNA diagnostic systems
1. Bind ssDNA(target) to membrane
2. Hybridize to labeled ssDNAor 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
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 thatcauses
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) and certain E.
coli (gastroenteritis).
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 inmicrosatellites
• Examples of such polymorphisms include VNTRs,
STRs,RFLPsandSNPs
– Variable number tandem repeats
– Short Tandem Repeats
– Restriction fragment length polymorphism
– Single Nucleotide Polymorphism
Uses of DNAfingerprinting
• 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, bombblast)
Preparation of a DNA fingerprint
Step 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

47
• DNA fingerprinting is a comparative process:
– DNA from crime scene is compared with DNA of a
suspect
– So minimum of two samples must beprepared
Step 2
• DNAextraction
– standardized methods have been developed
– need to separate DNA from other cellmaterial
and debris from crime scene.

48
Step 3

• PCRusing primers targeting STRsat different loci

• PCRamplify STRsusing target sites on
chromosome

49
 Step 3
PCRamplification of DNA

1 strand Heat to
of DNA denatur
e STR locus
double-
stranded
DNA

Design primers that anneal to STR locus STR locus

Amplify all the regions of the chromosome

where the STRs exist.
50
• Since the # of times sequence is repeated is
different for each person, fragment size willbe
different.
• This is done for 13 different STRsequences
• Differences occur among individuals at each of
the 13 loci on the chromosome where the STRs
occur
• This allows for a lot of variation

52
Restriction Fragment Length Polymorphism
For 1 STR sequence at 1 locus

Person A Forensic sample

STR STR

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…..

PCR amplify
STR region
C-C-X-X-G-G
C-C-X-X-X-G-G

well well

Gel
electrophoresis

53
 Banding Patterns
• 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

54
Some examples of DNA fingerprinting

• Paternity cases
• Crime scenes
Example

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

56
Technical Considerations

• Preserve the integrity of DNAsample

• Avoid DNA contamination & degradation
• Avoid incomplete digestions if REsareused
• Use standard hybridization conditions
• Use standard PCRprimers and procedures
• Gel analysis is less reproducible than capillary
electrophoresis of PCRproducts
Test Choice
• Cost
• Sample requirements
• Sensitivity/Specificity
• Positive/ Negative predictive value