Molecular diagnostics techniques analyze biological markers in the genome and proteome for disease diagnosis and personalized medicine. The history of these techniques includes pivotal developments from early genetic tests for thalassemia to the establishment of professional organizations. Key applications include pathogen detection, antibiotic resistance identification, and forensic uses, with techniques like plasmid profiling, nucleotide sequencing, and amplification methods being critical.

1. Molecular Diagnostics
Techniques
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2. Presented by:
SAYED MUHAMMAD TOUSEEF
Department of Biotechnology
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3. Outline
Introduction
History
How it works
Applications
Techniques used
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4. Introduction of
MOLECULAR DIAGNOSTICS
TECHNIQUES
 Molecular diagnostics is a collection of techniques.
 used to analyze biological markers in
the genome and proteome.
 The technique is used to diagnose and monitor disease,
detect risk, and decide which therapies will work best for
individual patients.
 molecular diagnostics offers the prospect
of personalized medicine.
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6. History
 In 1980, Yuet Wai Kan et al. suggested a prenatal genetic test
for Thalassemia that did not rely upon DNA sequencing—then in
its infancy—but on restriction enzymes that cut DNA where they
recognized specific short sequences, creating different lengths of
DNA strand depending on which allele (genetic variation) the fetus
possessed.
 in 1995, the Association for Molecular Pathology (AMP) was
formed to give it structure.
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7.  In 1999, the AMP co-founded The Journal of Medical
Diagnostics.
 In 2012, molecular diagnostic techniques for
Thalassemia use genetic hybridization tests to identify
the specific single nucleotide polymorphism causing an
individual's disease
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8. How it works??
 Every organisms contain some unique species specific DNA
sequences.
 Molecular diagnostics makes the specie specific DNA visible
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9. Applications
 Classification of organism based on genetic relatedness
(genotyping)
 Identification and confirmation of isolate obtained from
culture
 Early detection of pathogens in clinical specimen
 Rapid detection of antibiotic resistance
 Detection of mutations
 Detection of toxigenic from non toxigenic strains
 Also useful in forensic medicine
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10. Techniques used
 Plasmid profiling
 Nucleotide sequencing
 Restriction Fragment Length Polymorphism
(RFLP)
 Amplification techniques
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11. Plasmid profiling
 Plasmids are the extra chromosomal circular double stranded DNA
found in most bacteria
 Each bacterium has one or several plasmids
 Cells are lysed and the nucleic acid are subjected to electrophoresis
 The size and number of plasmids can be estimated
 Drawback: some species may contain variable number of plasmids or
even unrelated bacteria may have similar number of plasmids
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13. Nucleotide sequencing
 For determination of the nucleotide sequence in the given
DNA molecule
 Methods:
1. Chemical cleavage method
2 .Chain termination method
 Not much role in diagnostic microbiology
For structure of gene, mutations and to design primers
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14. Restriction Fragment Length
Polymorphism (RFLP)
 Polymorphism in nucleotide sequence is present in all
organism
 Restriction sites are the strands of DNA that are specifically
recognized and cleaved by restriction endonucleases
 Useful as a
1. Epidemiological typing tool
2. Ribotyping – phylogenetic classification
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16. Nucleic Acid Sequence Based
Amplification
• Isothermic non PCR procedure
• Definition: A primer dependent technology that
can be used for the continuous amplification of
nucleic acid in a single mixture at one
temperature.
• 3 SR: self sustained replication
• 3 enzymes: AMV reverse transcriptase,
Ribonuclease H, t7 RNA polymerase
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17. Transcription Based Amplification
• Useful in the amplification of ss RNA rather than
DNA
• Developed by Gen-probe
• Used in clinical laboratories to detect Chlamydia
trachomatis and Neisseria gonorrhea
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18. Strand Displacement Amplification
• It is an isothermal technique
• Based on restriction endonuclease nicking its
recognition site and a polymer extending the
nick at its 3’ end displacing the downstream
strand
• Required restriction enzyme cleavage of the
DNA sample prior to amplification
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19. cont…
• Normally restriction enzyme cleavage produce dsDNA
which is not suitable template for SDA
• By incorporating alpha thio substituted nucleotides a
double stranded hemiphosphorothioated DNA is created
where the restriction site in newly synthesized strand is
resistant to cleavage
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21. Signal Amplification
• Amplify the signal generated by the labelled
probes
• bDNA-Branched DNA probes
• Hybrid Capture- Anti DNA-RNA hybrid antibody
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22. Cont…
• Signal amplification-used to increase the sensitivity of the
probe based assays
• 103-105 nucleic acid targets can be detected
• Branched DNA probe system:
• Target sequence is captured using a capture step
• Hybridization with an unlabeled probe that has two
hybridization sequence
• One directed against target sequence
• Second hybridizes with bDNA amplification number
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23. Cont…
• Multimer system—chemically synthesized
oligonucleotide chain with a comb like backbone that can
bind to several reporter probes
• Highly sensitive because the target nucleic acid has to
bind both to the capture as well as target probes before
the signals are amplified
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24. THANK-YOU
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