The document discusses molecular diagnostics and genetic testing techniques. It provides an overview of molecular diagnostics, their significance in medicine, and how they are transforming fields like prenatal testing, disease detection, and drug selection. It then covers various immunological diagnostic methods like ELISA, radioimmunoassay, western blotting, and their characteristics. The document also discusses molecular genetic tests, genetic alterations detected, and techniques for DNA-based diagnosis of diseases. It focuses on the principles and procedures of molecular diagnostic methods like hybridization assays and PCR and their applications in detecting pathogens and genetic disorders.
Kolkata Call Girls Shobhabazar 💯Call Us 🔝 8005736733 🔝 💃 Top Class Call Gir...
Molecular diagnostics
1. CHC 812: CELL BIOLOGY, GENETICS,
IMMUNOLOGY AND TERATOLOGY
Lecture 01, 2015
Molecular Diagnostics
Lecturer: Dr. G. Kattam Maiyoh
GKM/M.MEDPAEDS/LECT 01/2015
2. 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.
GKM/M.MEDPAEDS/LECT 01/2015
3. Molecular Diagnostic
• USE OF:
– Sequence Specific INFORMATION
• in
– MACROMOLECULES
• for
– Risk identification
– Diagnosis
– Prognosis
– Prediction of response to therapy
– Monitoring therapeutic responses
GKM/M.MEDPAEDS/LECT 01/2015
5. 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.
GKM/M.MEDPAEDS/LECT 01/2015
6. 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
GKM/M.MEDPAEDS/LECT 01/2015
7. 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)
GKM/M.MEDPAEDS/LECT 01/2015
8. 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
GKM/M.MEDPAEDS/LECT 01/2015
10. Target antigens and polyclonal versus
monoclonal antibodies
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.
Target antigen
with various antigenic
determinants (epitopes)1
2 3 4
5
67
GKM/M.MEDPAEDS/LECT 01/2015
14. Enzyme-Linked Immunosorbent Assay (ELISA):
Immunological detection
Target molecule
antigenic site
iiiiiiiiiiiiiii
Support
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
Y
YY
Y
bound primary
antibody
YE
YE
YEYE
enzyme linked
secondary antibody
colorless substrate
colored product
GKM/M.MEDPAEDS/LECT 01/2015
15. 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!!!
GKM/M.MEDPAEDS/LECT 01/2015
16. ELISA -Variants
Detection based on
enzyme catalyzed
reactions:
1.alkaline Phosphatase
2.horseradish peroxidase
3. β-galactosidase
Detection based on
fluorescent labeled
secondary antibody
GKM/M.MEDPAEDS/LECT 01/2015
17. ELISA –Variants
The 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
GKM/M.MEDPAEDS/LECT 01/2015
19. 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.
GKM/M.MEDPAEDS/LECT 01/2015
23. 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
GKM/M.MEDPAEDS/LECT 01/2015
24. Immuno Electron Microscopy
electron-dense labels
absorb 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
25. We now know how God
wrote the book of life
Bill Clinton
30. 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.
GKM/M.MEDPAEDS/LECT 01/2015
32. 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
GKM/M.MEDPAEDS/LECT 01/2015
34. 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.
GKM/M.MEDPAEDS/LECT 01/2015
35. 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.
GKM/M.MEDPAEDS/LECT 01/2015
37. 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
38. Nonradioactive Hybridization Procedures
• Use of biotin-labeled nucleotides in DNA probes
instead of 32
P, 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)
GKM/M.MEDPAEDS/LECT 01/2015
41. 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
GKM/M.MEDPAEDS/LECT 01/2015
42. 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
GKM/M.MEDPAEDS/LECT 01/2015
43. 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
GKM/M.MEDPAEDS/LECT 01/2015
44. 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
GKM/M.MEDPAEDS/LECT 01/2015
45. 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
January 23, 2015 47
GKM/Forensic and Clinical Bioc./Lec
03/2013
46. • 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.
January 23, 2015 48
GKM/Forensic and Clinical Bioc./Lec
03/2013
47. Step 3
• PCR using primers targeting STRs at different loci
• PCR amplify STRs using target sites on
chromosome
January 23, 2015 49
GKM/Forensic and Clinical Bioc./Lec
03/2013
48. Step 3
PCR amplification of DNA
1 strand
of DNA
Heat to
denature
double-
stranded
DNA
Design primers that anneal to STR locus
Amplify all the regions of the chromosome
where the STRs exist.
STR locus
STR locus
January 23, 2015 50
GKM/Forensic and Clinical Bioc./Lec
03/2013
49. PCR allows you to
make millions of
copies of the STR
region from a single
copy of DNA you
recovered from crime
scene.
January 23, 2015 51
GKM/Forensic and Clinical Bioc./Lec
03/2013
50. • 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
January 23, 2015 52
GKM/Forensic and Clinical Bioc./Lec
03/2013
51. 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 amplify
STR region
STR
well well
Gel
electrophoresis
Person A Forensic sample
For 1 STR sequence at 1 locus
January 23, 2015 53
GKM/Forensic and Clinical Bioc./Lec
03/2013
52. • 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
January 23, 2015 54
GKM/Forensic and Clinical Bioc./Lec
03/2013
Banding Patterns
53. Some examples of DNA fingerprinting
• Paternity cases
• Crime scenes
GKM/M.MEDPAEDS/LECT 01/2015
57. 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
GKM/M.MEDPAEDS/LECT 01/2015
58. Test Choice
• Cost
• Sample requirements
• Turnaround time
• Sensitivity/Specificity
• Positive/ Negative predictive value
• Type of mutation detected
• Genotyping vs mutation scanning
GKM/M.MEDPAEDS/LECT 01/2015
59. 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?
GKM/M.MEDPAEDS/LECT 01/2015
60. • 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)
GKM/M.MEDPAEDS/LECT 01/2015
61. 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
GKM/M.MEDPAEDS/LECT 01/2015
62. 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
GKM/M.MEDPAEDS/LECT 01/2015