1. Polymerase Chain Reaction (PCR) and Its Applications

2. Applications of PCR Detection of pathogens Classification of organisms
DNA fingerprinting
Drug discovery
Genetic matching
Genetic engineering
Pre-natal diagnosis
Classification of organisms
Genotyping
Molecular archaeology
Mutagenesis
Mutation detection
Sequencing
Cancer research

3. Applications of PCR Basic Research Applied Research Mutation screening
Drug discovery
Classification of organisms
Genotyping
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
Bioinformatics
Genomic cloning
Site-directed mutagenesis
Gene expression studies
Genetic matching
Detection of pathogens
Pre-natal diagnosis
DNA fingerprinting
Gene therapy

4. Applications of PCR Molecular Identification Sequencing
Genetic Engineering
Bioinformatics
Genomic cloning
Human Genome Project
Site-directed mutagenesis
Gene expression studies
Molecular Archaeology
Molecular Epidemiology
Molecular Ecology
DNA fingerprinting
Classification of organisms
Genotyping
Pre-natal diagnosis
Mutation screening
Drug discovery
Genetic matching
Detection of pathogens

5. MOLECULAR IDENTIFICATION:

7. Detection of Unknown Mutations
Molecular Identification:
Detection of Unknown Mutations

8. SSCP gels: “shifts” representing a mutation in the amplified DNA fragment

9. Classification of Organisms
Molecular Identification:
Classification of Organisms
Insufficient data
1) Relating to each other
2) Similarities
3) Differences
* Fossils
* Trace amounts
* Small organisms
! DNA !

11. Rademaker et al. 2001

12. Detection Of Pathogens
Molecular Identification:
Detection Of Pathogens

13. Detection Of Pathogens
Molecular Identification:
Detection Of Pathogens
Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

14. Genotyping by STR markers
Molecular Identification:
Genotyping by STR markers

15. Prenatal Diagnosis Chorionic Villus Amniotic Fluid
Molecular Identification:
Prenatal Diagnosis
644 bp
440 bp
204 bp
Chorionic Villus
Amniotic Fluid
Molecular analysis of a family with an autosomal recessive disease.

16. SEQUENCING radioactive fluorescent
Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP)
NO polymerisation after a dideoxynucleotide!
Fragments of DNA differing only by one nucleotide are generated
radioactive
Nucleotides are either or
fluorescent

18. Classical Sequencing Gel

19. Reading Classical Sequencing Gels

20. Sequencing:

21. Applied Biosystems 310 Genetic Analyzer
6/14/2018
Applied Biosystems 310 Genetic Analyzer

22. The Process In a Nutshell
6/14/2018
The Process In a Nutshell
Amplified DNA samples are injected into a capillary. Fluorescent tags on the DNA fragments are excited by a laser as they pass a window in the capillary, the fluorescence is recorded by a camera, and this signal is converted into a “peak” by the computer software.

23. 6/14/2018
STR data
X, Y,
X Y

24. 6/14/2018
STR data (cont’d)

25. 6/14/2018
STR data (cont’d)
“The DNA profile obtained from Item 25(S) matches the DNA profile of the suspect. The combination of genetic marker types exhibited by Item 25(S) and the suspect occurs in approximately one in one hundred quadrillion (1017) individuals…”

26. Summary
blood, chorionic villus, amniotic fluid, semen, hair root, saliva
68,719,476,736 copies
Gel Analysis, Restriction Digestion, Sequencing

27. Conclusion The speed and ease of use, sensitivity, specificity and
robustness of PCR has revolutionised molecular biology
and made PCR the most widely used and powerful
technique with great spectrum of research and
diagnostic applications.