Molecular techniques are used to analyze biological markers in genomes and proteomes. They provide several advantages over traditional diagnostic methods such as faster diagnosis, increased sensitivity and specificity, and ability to detect pathogens more rapidly. Common molecular techniques include PCR, real-time PCR, nucleic acid sequencing, microarrays, and nucleic acid amplification methods like NASBA. These techniques are useful for diagnosing infectious diseases and genetic conditions.

1. Molecular Techniques For
Disease Diagnosis
PRIYANKA GUPTA
PhD Research Scholar
Dept. Of Lab. Medicine , AIIMS,
New Delhi

2. Molecular Techniques
 Molecular techniques is a
collection of techniques used
to analyse biological markers
in the genome and proteome.
 Several molecular applications
have been incorporated in the
routine diagnostic labs which
are more user-friendly, cost-
effective, and accurate profile.

3. Why go for molecular way?
Traditional methods pose several challenges
• Growth of fastidious pathogens
• Maintenance of viability
• Delay in cultivation
• Non-culturability of certain organisms.
• Hazardous to propagate in lab.
• Cost versus clinical utility.

4. Advantage of Molecular Methods
 Aid in faster diagnosis of Diseases.
 Increased sensitivity and specificity.
 Rapid detection of pathogen than conventional
methods.
 Decrease the man power need for detection.
 Give rapid answers to treatment options in life
threatening diseases.
 Adapted to instrumentation.

5. Family Tree of Molecular Techniques

6. Types of Molecular Techniques
 Gel Electrophoresis
 Agarose gel elecrophoresis
 Polyacrylamide gel
electrophoresis
 Molecular Hybridization
 Southern blot
 Northern Blot
 Western Blot
 Dot blot assay
 Fluorescence In situ
hybridization (FISH)
 Mutation detection
 RFLP
 RAPD
 DNA Sequencing
Automated
Pyrosequencing
Next generation sequencing
 Nucleic Acid Amplification Based
Techniques
 PCR
 qPCR
TMA
NASBA
SDA
LAMP
LCR
DNA Microarray

7. Nucleic acid amplification based
Techniques
 PCR
 qPCR
 TMA
 NASBA
 SDA
 LAMP
Signal based
amplification
Probe based
amplification
Target based
amplification
LCR – Ligase
Chain Reaction
 bDNA –
Branched DNA
probes

8. Target Based Amplification
 In-vitro systems for enzymatic replication of target
molecule to detectable levels.
 Allows target to be identified and further
characterized.
 Examples: Polymerase chain reaction, transcription
mediated amplification, strand displacement
amplification and nucleic acid sequence-based
amplification etc.

9. Polymerase Chain Reaction (PCR)
 Karry Mullis (1983)
 PCR is first nucleic acid amplification method used
for selective amplification of target sequence.
 The method relies on thermal cycling, consisting of
cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication
of the DNA.
 Types of PCR: PCR, Nested PCR, RT PCR, qPCR,
Multiplex PCR, etc.

10. PCR
Requirement
 DNA template
 Primers
 Taq Polymerase
 Deoxynucleotide
 Buffer solution
 Divalent cations
(Magnesium or
manganese ions
Steps

11. DETECTION OF PCR PRODUCT
(Post amplification analysis)
 Gel electrophoresis is the most common method

12. Advantages
• Speed
• Ease of use
• Versatile technique
Limitations:
• Need for target DNA sequence information
• Need of expensive equipment.
• Lack of proof reading activity in Taq polymerase (Error
40% after 20 cycles)
• Short size and limiting amounts of PCR product

13. Application:
• Diagnosis of genetic disease.
• e.g. Sickle cell anaemia, haemophilia, ß-thalassemia,
etc.
• PCR permits early diagnosis of malignant disease.
Such as leukaemia & lymphoma.
• PCR use for detection of noncultivatable or slow
growing microorganism like mycobacteria, anaerobic
bacteria & viruses e.g. Hepatitis, HIV, Influenza virus
infection.

14. Real time PCR (qPCR)
• DNA amplification analysis is monitored
simultaneously over the course of thermocycling.
The amplification product is detected as it
accumulate.
• Real time PCR monitors the fluorescence emitted
during the reaction as an indicator of amplicon
production during each PCR cycle.

15. • In real time PCR two different types methods are used for
detection :
• Method -1:-Real-time PCR using double-stranded DNA
dyes. (e.g. SYBR Green)
• Method -2:-Fluorescent reporter probe method
Advantages
• Collect data in the exponential growth phase.
• An increase of reporter fluorescent signal is directly
proportional to the number of amplicons generated.
• No post PCR processing

16. Limitations:
• Requires expensive equipments and reagents
• High technical skill & support.
• DNA contamination
Application:
 Pathogen detection
 Genotyping
 Gene expression analysis
 SNP allelic discrimination
 Mutation detection
 Viral load quantification.

17. Transcription Mediated Amplification
Techniques
 Both are isothermal RNA amplifications modeled after
retroviral replication
 RNA target is reverse transcribed into cDNA, followed by
RNA synthesis via RNA polymerase
 Amplification involves synthesis of cDNA from RNA
target with a primer containing the T7 RNA pol promoter
sequence
Transcription mediated
amplification (TMA)
Nucleic acid sequence based
amplification (NASBA)

18. • TMA is an RNA transcription amplification system using two
enzyme to drive the reaction : RNA polymerase and reverse
transcriptase.
• TMA is isothermal, the entire reaction is performed at the same
temperature is a water bath or heat block. This is in contrast to
other amplification reactions such as PCR or LCR that require
a thermal cycler.
• TMA can amplify either DNA or RNA and produces RNA
amplicon, in contrast to most other nucleic acid amplification
methods that only produce DNA.
• TMA has very rapid kinetics resulting in a billion fold
amplification with in 15-30 minutes.
Transcription mediated amplification
(TMA)

20. Application:
• TMA have been used for diagnosis of different
infectious diseases caused by:
• HIV
• Hepatitis viruses
• Chlamydia trachomatis
• Neisseria gonorrhoeae
• Mycobacterium tuberculosis.

21. Nucleic Acid Sequence Based
Amplification (NASBA)
 Compton (1991)
 Also called Self sustained sequence based
amplification.
 It is a sensitive, isothermal, transcription based
amplification system specially used for RNA targets
like m-RNA, r-RNA or genomic RNA.
 It is very similar to TMA.
 This is more complex than PCR. But its does not
require thermal cycling.
 It is more sensitive than to reverse transcriptase PCR

22. Characteristics of NASBA
 It based on three enzymes activity:
 Avian myloblastosis virus reverse transcriptase
(AMV-RT).
 RNase H
 T7 DNA dependent RNA polymerase.
 This technique work on homogenous isothermal
temperature 41°C for 90 to 120 minutes.

23. viral RNA strands (Sense)
Primer P1 binds to the RNA and is
elongated by reverse transcriptase
(AMV-RT).
DNA : RNA hybrid
RNA hydrolyzed by RNase H.
primer P2 can also bind ,
a dsDNA molecule synthesize.
Primer P1 is designed in such a
manner that when it forms a double-
stranded DNA, it codes for a T7
RNA polymerse Promoter site.
Generate antisense RNA copies
using a DNA template.
The new copies of DNA are
generated using RNA.
Here in this case, P2 will bind first.
STEPS Of NASBA

24. Advantage:
 Very less time taken method 90minuts.
 Chance very less for contamination.
Disadvantage:
 Enzyme are not thermostable.
 Complex steps.

25. • Rapid diagnostic tests for several pathogenic viruses
and bacteria:
– Influenza A
– Foot –and mouth disease virus
– Severe acute respiratory syndrome (SARS)-associated
coronovirus.
– Mycobacteria tuberculosis
– HIV
– Hepatitis C viruses, etc.
Application:

26. Strand Displacement Amplification(SDA)
 This process consists of two phase-Target generation
and second is amplification.
 This process requires:
 Two primer pairs (B1,S1 and B2,S2).
 Two enzymes pairs( DNA polymerase and a
restriction enzyme).
 dATP and dAMP.
 This process is efficient for small targets of less than
200 bp.

27. Steps of SDA
Denaturation of DNA
attachment of the primers.
Elongation of B1 and B2 primer to
produce the target DNA
Primer S1 and S2(having nick able
site) binds with the target DNA and
elongation takes place
Incorporation of dAMP into the
target DNA at the nick able site .
Nicking of the DNA
strand(produced from primer s1
and s2) takes place and that DNA
strands are separate out.
DNA polymerase again act at the
nick able site and extends that
strand again and the cycle
continues.

28. Loop Mediated Isothermal Amplification
(LAMP)
• Loop mediated isothermal amplification is an
advance powerful innovative technology used
for diagnosis of microbial diseases in very
rapid & simple way.
• This technique different to PCR on the bases
of temperature & Bst DNA polymerase (Bst-
Bacillus stearothermophylus)
• LAMP has an improved simple visual
amplicon detection system.

29.  Use 6 specially designed primers spanning 8 distinct
regions on the target gene.
 The amplification proceeds at a constant temperature
60-65° C for 45 -60 minutesusing strand
displacement reaction.
 Amount of DNA produce by LAMP is high as
compare to PCR.
 Mechanism of LAMP includes three steps:
 Production of Starting Material.
 Cycling amplification
 Elongation & recycling.

30. Steps of LAMP :

31.  Use simple method for
detection:
 Photometer: Detection based
of turbidity of sample
 Fluorescence dye : SYBR
Green, SYTO 9
Detection methods:

32. Applications:
• Simple and easy screening assay.
• Useful for diagnosis of infectious diseases such as:
– Tuberculosis
– Maleria
– Kala azar
– Hepatitis, etc
Limitation:
 Less versatile than PCR.
 Not very useful for multiplexing as compared to
PCR

33. Probe Based Amplification
Ligase Chain Reaction
 Amplifies the nucleic acid used as a probe.
 Two probes are used per each DNA strand and
are ligated together to form a single probe.
 Uses both a DNA polymerase enzyme and a
DNA ligase enzyme to drive the reaction.
 Requires thermo cycler.
 Each cycle results in doubling of target.
 Greater specificity than PCR.

34. • Denaturation of target DNA at 94c.
• Annealing of two pairs of synthetic deoxyoligomeres
at40-60c.
• Members of each pair bind in such a way that they are
immediately adjacent and completely cover the target
sequence on both separated DNA strands.
• The oligomeres of each pair is then joined by a
thermostable DNA ligase.
• This doubles the target DNA molecules and complete
the first LCR cycle
• The second cycle is initiated by denaturation.
STEPS OF LIGASE CHAIN REACTION

36. Application
 Detection of single base mutations in genetic
diseases.
 Neisseria gonorrhoea
 Chlamydia tracomatis

37. Signal Based Amplification
• Replicates signal rather than either the target or the probe.
Branched DNA assay:
• A branched DNA assay begins with a dish or some other
solid support (e.g., a plastic dipstick).
• The dish is coated with small, single stranded DNA
molecules (or chains) that 'stick up' into the air. These are
known as capture probe DNA molecules.
• Next, an extender DNA molecule is added. Each extender
has two domains, one that hybridizes to the capture DNA
molecule and one that "hangs out" in the air.

38.  The purpose of the extender is two fold:
 First, it creates more available surface area for target
DNA molecules to bind.
 Second, it allows the assay to be easily adapted to
detect a variety of target DNA molecules.
 Once the capture and extender molecules are in place
and they have hybridized, the sample can be added.
 Target molecules in the sample will bind to the extender
molecule. So we have a base peppered with capture
probes, which are hybridized to extender probes, which
in turn are hybridized to target molecules.

39. • At this point, signal amplification takes place.
• A label extender DNA molecule is added that has two
domains (similar to the first extender). The label extender
hybridizes to the target and to a preamplified molecule.
• The preamplifier molecule has two domains. First, it
binds to the label extender and second, it binds to the
amplifier molecule.
• An example amplifier molecule is an oligonucleotide
chain bound to the enzyme alkaline phosphatase.
• Branched chains are well suited to detection of nucleic
acid targets with sequence heterogeneity such as
hepatitis C and HIV.

41. Drawbacks of Molecular Techniques
 Potential for false-positive results due to contaminating
nucleic acids.
 PCR and LCR, DNA product main source of
contamination.
 TMA & NASBA RNA products are possible
contaminants.
 Must have product inactivation areas from amplification
areas and use of inactivation systems such as UV light
help alleviate contamination.
 Very expensive.

42. Future of Molecular Diagnostic
techniques
• Despite expense may be times that rapid diagnosis will
result in decreased cost.
• Example: Mycobacterium quick diagnosis no need for
expensive respiratory isolation.
• Incredibly useful in serology and microbiology.
• Increased specificity and sensitivity of molecular testing.
• Testing will continue to become more rapid as assays are
automated which will also bring down the costs.