Latest -PCR APPLICATION
by
A.Arputha Selvaraj
What is PCR?What is PCR?
It was invented in 1983 by Dr. Kary Mullis,
for which he received the Nobel Prize in
Chemistry in...
What is PCR? :What is PCR? :
Why “Polymerase”?Why “Polymerase”?
It is called “polymerase” because the only
enzyme used in ...
What is PCR? :What is PCR? :
Why “Chain”?Why “Chain”?
It is called “chain” because the products
of the first reaction beco...
What is PCR? :What is PCR? :
The “Reaction” ComponentsThe “Reaction” Components
1) Target DNA - contains the sequence to b...
The ReactionThe Reaction
THERMOCYCLERPCR tube
Denature (heat to 95o
C)
Lower temperature to 56o
C
Anneal with primers
Increase temperature to 72o
C
DNA polymerase + dNT...
DNA copies vs Cycle number
0
500000
1000000
1500000
2000000
2500000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2...
Applications of PCRApplications of PCR
• Classification of
organisms
• Genotyping
• Molecular
archaeology
• Mutagenesis
• ...
Applications of PCR
Basic Research Applied Research
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DN...
Applications of PCR
Molecular Identification Sequencing Genetic Engineering
• Molecular Archaeology
• Molecular Epidemiolo...
MMOLECULAROLECULAR IIDENTIFICATION:DENTIFICATION:
Detection of Unknown MutationsDetection of Unknown Mutations
Molecular Identification:
SSCP gels:
“shifts” representing a mutation
in the amplified DNA fragment
Classification of OrganismsClassification of Organisms
1) Relating to each other
2) Similarities
3) Differences
* Fossils
...
Rademaker et al. 2001
Detection Of PathogensDetection Of Pathogens
Molecular Identification:
Detection Of PathogensDetection Of Pathogens
Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.19...
Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)
GenotypingGenotyping by STR markersby STR markers
Molecular Identification:
Prenatal DiagnosisPrenatal Diagnosis
644 bp
440 bp
204 bp
Molecular analysis of a family with an autosomal recessive disea...
SSEQUENCINGEQUENCING
Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP)
NO polymerisation after a dideoxynucleot...
Classical Sequencing GelClassical Sequencing Gel
Sequencing:
Reading Classical Sequencing GelsReading Classical Sequencing Gels
Sequencing:
Sequencing:
SummarySummary
blood, chorionic villus,
amniotic fluid, semen,
hair root, saliva
68,719,476,736 copies Gel Analysis,
Restr...
ConclusionConclusion
The speedspeed and easeease of use, sensitivitysensitivity, specificityspecificity and
robustnessrobu...
Thank You
PCR APPLICATION & TECHNIQUES
PCR APPLICATION & TECHNIQUES
PCR APPLICATION & TECHNIQUES
PCR APPLICATION & TECHNIQUES
PCR APPLICATION & TECHNIQUES
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PCR APPLICATION & TECHNIQUES

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PCR APPLICATION & TECHNIQUES

  1. 1. Latest -PCR APPLICATION by A.Arputha Selvaraj
  2. 2. What is PCR?What is PCR? It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993. PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.
  3. 3. What is PCR? :What is PCR? : Why “Polymerase”?Why “Polymerase”? It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.
  4. 4. What is PCR? :What is PCR? : Why “Chain”?Why “Chain”? It is called “chain” because the products of the first reaction become substrates of the following one, and so on.
  5. 5. What is PCR? :What is PCR? : The “Reaction” ComponentsThe “Reaction” Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme
  6. 6. The ReactionThe Reaction THERMOCYCLERPCR tube
  7. 7. Denature (heat to 95o C) Lower temperature to 56o C Anneal with primers Increase temperature to 72o C DNA polymerase + dNTPs
  8. 8. DNA copies vs Cycle number 0 500000 1000000 1500000 2000000 2500000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Cycle number DNAcopies
  9. 9. Applications of PCRApplications of PCR • Classification of organisms • Genotyping • Molecular archaeology • Mutagenesis • Mutation detection • Sequencing • Cancer research • Detection of pathogens • DNA fingerprinting • Drug discovery • Genetic matching • Genetic engineering • Pre-natal diagnosis
  10. 10. Applications of PCR Basic Research Applied Research • Genetic matching • Detection of pathogens • Pre-natal diagnosis • DNA fingerprinting • Gene therapy • Mutation screening • Drug discovery • Classification of organisms • Genotyping • Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • Bioinformatics • Genomic cloning • Site-directed mutagenesis • Gene expression studies
  11. 11. Applications of PCR Molecular Identification Sequencing Genetic Engineering • Molecular Archaeology • Molecular Epidemiology • Molecular Ecology • DNA fingerprinting • Classification of organisms • Genotyping • Pre-natal diagnosis • Mutation screening • Drug discovery • Genetic matching • Detection of pathogens • Bioinformatics • Genomic cloning • Human Genome Project • Site-directed mutagenesis • Gene expression studies
  12. 12. MMOLECULAROLECULAR IIDENTIFICATION:DENTIFICATION:
  13. 13. Detection of Unknown MutationsDetection of Unknown Mutations Molecular Identification:
  14. 14. SSCP gels: “shifts” representing a mutation in the amplified DNA fragment
  15. 15. Classification of OrganismsClassification of Organisms 1) Relating to each other 2) Similarities 3) Differences * Fossils * Trace amounts * Small organisms ! DNA ! Molecular Identification: Insufficient data
  16. 16. Rademaker et al. 2001
  17. 17. Detection Of PathogensDetection Of Pathogens Molecular Identification:
  18. 18. Detection Of PathogensDetection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994) Molecular Identification:
  19. 19. Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)
  20. 20. GenotypingGenotyping by STR markersby STR markers Molecular Identification:
  21. 21. Prenatal DiagnosisPrenatal Diagnosis 644 bp 440 bp 204 bp Molecular analysis of a family with an autosomal recessive disease. Molecular Identification: • Chorionic Villus • Amniotic Fluid
  22. 22. SSEQUENCINGEQUENCING Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide! Fragments of DNA differing only by one nucleotide are generated Nucleotides are either or
  23. 23. Classical Sequencing GelClassical Sequencing Gel Sequencing:
  24. 24. Reading Classical Sequencing GelsReading Classical Sequencing Gels Sequencing:
  25. 25. Sequencing:
  26. 26. SummarySummary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction Digestion, Sequencing
  27. 27. ConclusionConclusion The speedspeed and easeease of use, sensitivitysensitivity, specificityspecificity and robustnessrobustness of PCR has revolutionised molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.
  28. 28. Thank You

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