What is the process of a molecular diagnostics

of infectious diseases?

• What is the purpose of a molecular diagnostics?

-Detection of nucleic acids of a specific organisms
(Bacteria/ virus)
• How to detect nucleic acids of an organism?
- Decide the specimen which contains
organism/nucleic acids
- Extract nucleic acid from specimens
• What is the method which can detect the presence of
nucleic acids?
- Design primers/ probes
- Select method
 For human genetic diseases

• Detect the causative gene

• Identify the positions of genes/mutations
cause of diseases
• Select a method for detection of the
mutations/ SNP/chromosome translocation
• Hybridization
• Amplification technology
- Quantitive PCR
- Qualitive PCR
• DNA sequencing
- Chemical Cleavage Sequencing
- Dideoxy Chain Terminator Sequencing
- Pyrosequencing
 The basic principles involved in
molecular hybridization

• based on the principles of denaturation and

renaturation of nucleic acids
• can be used as the qualitative and quantitative
approaches for the analysis of DNA or RNA.
• During the heat or alkaline denaturation, the dsDNA
becomes ssDNAs. The ssDNAs can renature during
the annealing.
• Either homogeneous or heterogeneous DNA or RNA
can form a hybridization molecule if they share the
complementary bases
• Southern Blot
• Northern Blot
• F.I.S.H (Fluorescence In Situ
Hybridization)
• CGH ( Comparative genomic
hybridization)
• Microarray
• FISH - a process which vividly paints
chromosomes or portions of chromosomes with
fluorescent molecules
• Opening picture - Human M-phase spread using
DAPI stain
• is a very sensitive technique for mapping genes
and identifying chromosomal abnormalities
(insertion, deletion, translocation).
• Used to identify the presence and location of a
region of DNA or RNA within morphologically
preserved chromosome preparations, fixed cells
or tissue sections
• In this process, the fluorescence labeled probe
is added to the metaphase spread of
chromosomes on a glass slide, and the exact
location of hybridization is observed under
fluorescent microscope.
• Aids in gene mapping, toxicological studies,
analysis of chromosome structural aberrations,
and ploidy determination
• Advantage: less labor-intensive method for
confirming the presence of a DNA segment
within an entire genome than other conventional
methods like Southern blotting,
 FISH Procedure

• Denature the chromosomes

• Denature the probe
• Hybridization
• Fluorescence staining
• Examine slides or store in the
dark
• Can detect multiple targets in a single
reaction
• Amplification of different regions in the
genome
• Test for multiple pathogens in a single
specimen
- Drug Resistance screen
- Herpes virus screen of CSF
PCR reactions can be devised in which several
targets are amplified simultaneously
 82oC 86oC 87.3oC 89.8oC
A B bp
60
LD 400
Fluorescence

BA 300
40 YP
200 LD (163 bp)

human HS (141 bp)

20 YP (121 bp)
100
BA ( 99 bp)

75 80 85 90 95
Temperature oC
• Pros:
Great for high throughput assays
Saves time, once established
Great for limited and valuable samples
aDNA, population genetic sample
• Cons:
Optimization can be time consuming
Product amplifications not equal
Primer design is a limiting step.
Cross dimming, controlling Tm
• Primer design
• Testing single primers
• Combining primers
• Optimization
– troubleshooting
• The PCR analysis of RNA.
• The RNA is isolated
• mRNA is converted to the first strand of
cDNA by a reverse transcription reaction
• The synthetic cDNA is then used as the
template in a conventional PCR.

Note: cDNA: complement DNA

 AAAAA Oligo dT primer is
RT TTTTT bound to mRNA

Reverse
AAAAA transcriptase
RT (RT) copies first
TTTTT
cDNA strand

Reverse
AAAAA RT
transcriptase
TTTTT
digests and
displaces mRNA
and copies
second strand of
cDNA

Double
strand
cDNA

Conversion of mRNA to cDNA by Reverse Transcription

• Ability to identify rare or low levels of mRNA
transcripts with great sensitivity.
• Especially useful when detecting viral gene
expression and the differentiation between
the latent and active virus.
• The gene cloning becomes much easier
because the target gene can be obtained
from trace RNA without the construction of a
cDNA library for target gene screening
BEACON
SCORPION
Taqman method
Genotyping result
 Real time PCR Applications
■ Pathogen detection
■ Viral quantification
■ Quantitation of gene expression
■ Genotyping
■ Drug Therapy Efficacy
■ DNA Damage measurement
■ Microarray Verification
Tetra-primer ARMS-PCR
Allele-specific PCR amplification
(ARMS_PCR)
aa aa a c a c
 Invader® Technology (Third Wave, Inc.)
• Directly detects specific nucleic acid sequences
• Based on signal amplification
• Isothermal reactions; no thermal cycling
• Method
- An overlapping structure created with the mutant
probe and the Invader® oligo. Cleavase® enzymes
cleave the primary probes that form overlapping
structures releasing the 5' flaps plus one nucleotide.
- Released flaps from the primary reaction serve as
oligos in a second simultaneous reaction on a labeled,
synthetic oligo (Fluorescence resonanceenergy
transfer (FRET) probe).
- Cleavage of this FRET probe = fluorescent signal.
• Produce 1 million to 10 million labeled cleavage
products per target sequence
MICROSATELITE
Fragment analysis
 PCR with 1 labeled and 1
unlabeled primer
 denaturing high-performance liquid
chromatography (dHPLC)

Column retention time

 Single Strand Conformational Polymorphism

PCR product is denatured,

chilled quickly and
run on non-denaturing gels.

Any sequence change

causes
formation a different
conformer
resulting in variation in
strand mobility.

Needs sequencing to prove

a significance of the change
 A differences in conformation
depends on conditions
of gel running
(% of buffer and acrylamide,
glycine in buffer,
temperature etc….)

Often it is important
to check 3-5 different conditions

before undoubtful resolution

can be achieved

Very cheap, 80% sensitive;

short sequences only
 Minisequencing by primer extension

DNA polymerase + one of the four labeled ddNTPs

= sequencing of one nucleotide
• PYROSEQUENCING is a unique method
for short-read DNA sequencing and
single nucleotide sequence variation
analysis
 Principle of Pyrosequencing

Step 1
A sequencing primer is hybridized to a single
stranded PCR amplified DNA template,

and incubated with the enzymes:

-- DNA polymerase,
-- ATP sulfurylase,
-- luciferase
-- apyrase,

and the substrates:

-- adenosine 5´ phosphosulfate (APS)
-- luciferin.
 Principle of Pyrosequencing
Step 2
The first of four dNTP is added to the reaction.
DNA polymerase catalyzes the incorporation of the
dNTP into the DNA strand, if it is complementary to
the base in the template strand.
Each incorporation event is accompanied by release
of an equimolar quantity of pyrophosphate (PPi)
 Principle of Pyrosequencing

Step 3
ATP sulfurylase
PPi  ATP.
ATP used in
the conversion of luciferin
to oxyluciferin
(visible light
proportional to ATP production).
The light is detected
by a charge coupled device
(CCD) camera
and seen as a peak in a pyrogram™.
Light signal is proportional to the number of nucleotides
incorporated.
 Principle of Pyrosequencing
• Step 4
Apyrase, a nucleotide degrading enzyme,
continuously degrades unincorporated dNTPs and
excess ATP. When degradation is complete, another
dNTP is added.
 Principle of Pyrosequencing
Addition of dNTPs is performed one at a time.

thio triphosphate
(dATP-alphaS)
is used as a substitute
for the natural
(dATP), as natural dATP
is not good for luciferase
 Chemistry/Demultiplexing/Detection Options in SNP Genotyping
Enzyme Chemistry Demultiplexing Detection Method Platform/Company
Illumina
BeadArrayTM

Allele-Specific Semi-Homogen. Luminex 100 Flow

Extend + Ligate Cytometry

Sequenom iPlexTM
Oligonucleotide Solid phase Mass Spec.
Ligation Assay microspheres Fluorescence

ABI SNPlexTM
Single Nucleotide
Primer Extension Homogeneous Mass Microarray
Spectrometry Minisequencing

ABI TaqmanTM
Capillary 5’-Nuclease
Allele-Specific Electrophoresis
Hybridization Fluor Res Energy
Transfer-FRET ABI SNaPShotTM

Solid phase
microarray “DASH”,
Amplicon Tm
Allele-Specific Fluorescence
PCR Polarization Perkin-Elmer
FP-TDI