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Polymerase chain reaction
Presented by: Aqsa Abbas
(2016-bt-032)
Submitted to: Dr Ali Raza Awan
List of Contents
• Introduction to PCR
• Brief History
• Basic principle
• components
• Steps of PCR
• Detection of PCR Product
• Applications
• errors and Disadvantages
• Conclusion
Introduction to PCR
• Polymerase chain reaction creates millions of copies of a
specific gene or any piece of DNA quickly in the test tube.
• PCR takes its name from the DNA polymerase, the
enzyme that carries out the DNA replication in the cell.
• it is considered a chain reaction because DNA
polymerase will carry out the replication over ad over
again till the millions of copies of desired gene are
formed.
Brief History of PCR
• 1983: Dr. Kary Mullis developed PCRM
• 1990: amplification and detection of specificDNA sequences
using afluorescent DNA-binding dye, laying the foundation
for future "real-time" or "kinetic" PCR.P
• 1991: RT-PCRis developed using asingle thermostable
polymerase, rTth, facilitating diagnostic tests for RNA
viruses.
• 1993:Dr. Kary Mullis shares Nobel Prize in Chemistry
for conceiving PCRtechnology.
Basic Principle
• PCR involves the Invitro primer mediated enzymatic Amplification
of the DNA.
• Polymerase chain reaction (PCR) is a technology used for quick
and easy amplifying DNA sequences, which is based on the
principleof enzymatic replicationof the nucleic acids.
• Its purpose is to amplify a lot of double-stranded DNAmolecules
(fragments) with same (identical) size and sequence by
enzymatic method and cyclingconditions.
Components of PCR
• DNA template (the sample DNA that contains the target
sequence to amplify)
• Deoxyribonucleoside triphosphates (dNTPs)
• PCR buffer
• Primers (forward and reverse)
• Taq polymerase
Steps of polymerase chain reaction-PCR
• To perform PCR, extracted sample (target DNA template)
is added to a tube containing primers, free nucleotides
(dNTPs), and Taq polymerase.
• The PCR mixture is placed in a PCR machine.
• PCR machine increases and decreases the temperature
of the PCR mixture in automatic, programmed steps
which generates copies of the target sequence
exponentially.
PCR STEPS
• Initial Denaturation:
– Performed only once at the start of PCR
– Usually at 95°C for 3-5 min.
– Ensures melting of genomic double-stranded DNA.
•
• Cycling Reactions
– Denaturation
• usually at 94°C for 30-45 sec
• double strand melts (opens) to single stranded DNA.
• All enzymatic reactions stop.
– Annealing
• Most variable step: 54-62°C for 20-45 sec
• Primers that fit exactly on double-stranded template DNA bind tighter.
– Extension
• Taq DNA polymerase optimal temperature: 70-72°C.
• Taq DNA polymerase adds dNTP in the 5'→3' direction.
–
• Final Extension
– Varies from 5-10 min.
– All single-stranded DNA is double-stranded.
This is how
PCR works...
PCR Cycle - Step 1
Target Sequence
Target Sequence
Denaturation by Heat
PCR Cycle - Step 2
Primers Anneal to Ends of Target
PCR Cycle - Step 3
Taq DNA Polymerase Catalyses primer
extension as the nucleotides are added.
Primer Extension as Nucleotides are
Added
End of the 1st PCR Cycle
Two Copies of Target Sequence
Target Amplification
No. of No. Amplicon
Cycles Copies of
1 2
2 4
3 8
4 16
5 32
6 64
20 1,048,576
30 1,073741824
1 cycle = 2 Amplicon
2 cycle = 4 Amplicon
3 cycle = 8 Amplicon
4 cycle = 16 Amplicon
5 cycle = 32 Amplicon
6 cycle = 64 Amplicon
7 cycle = 128 Amplicon
Detection of PCR products
• Labeled probe that is specific for the target gene
sequence is used to detect PCR amplified gene product
(also known as amplicon). Based on the nature of the
reporter molecule used, probe generates radioactive,
colorimetric, fluorometric, or chemiluminescent signals.
Probe based detection of amplicons serves two
purposes:
• It allows visualization of the PCR product
• It provides specificity by ensuring that the amplicon
is the target sequence of interest and not the result
of non-specific amplification.
• Apart from DNA based hybridization method,
sometimes simple gel electrophoresis method is
sufficient to confirm the presence of specific
amplicons.
Applications of PCR
• Identification and characterization of infectious agents
• Direct detection of microorganisms in patient specimens
• Detection of antimicrobial resistance
• Investigation of strain relatedness of pathogen of interest
• Genetic fingerprinting (forensic application/paternity
testing)
• Detection of mutation ( investigation of genetic diseases)
• Cloning genes
• PCR sequencing
Sources of errors
• Mispriming: Primer binds to off target sequence.
• Secondary DNA structure can obstruct primer
recognition.
• Primer dimerize and Amplify (Primer Dimer
formation).
• Disadventages of PCR include the problems with
the post PCR contamination due to the high
senstivity (false positive problem except for RT-
PCR).
conclusion
• PCR is not only vital in the clinical laboratory by
amplifying small amounts of DNA for STD detection,
but it is also important for genetic predisposing for
defects such asFactor V Leiden.
• The PCR technology can also be employed in law
enforcement, genetic testing of animal stocks and
vegetable hybrids, and drug screening along with
many more areas.
Polymerase chain reaction

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Polymerase chain reaction

  • 1. Polymerase chain reaction Presented by: Aqsa Abbas (2016-bt-032) Submitted to: Dr Ali Raza Awan
  • 2. List of Contents • Introduction to PCR • Brief History • Basic principle • components • Steps of PCR • Detection of PCR Product • Applications • errors and Disadvantages • Conclusion
  • 3. Introduction to PCR • Polymerase chain reaction creates millions of copies of a specific gene or any piece of DNA quickly in the test tube. • PCR takes its name from the DNA polymerase, the enzyme that carries out the DNA replication in the cell. • it is considered a chain reaction because DNA polymerase will carry out the replication over ad over again till the millions of copies of desired gene are formed.
  • 4. Brief History of PCR • 1983: Dr. Kary Mullis developed PCRM • 1990: amplification and detection of specificDNA sequences using afluorescent DNA-binding dye, laying the foundation for future "real-time" or "kinetic" PCR.P • 1991: RT-PCRis developed using asingle thermostable polymerase, rTth, facilitating diagnostic tests for RNA viruses. • 1993:Dr. Kary Mullis shares Nobel Prize in Chemistry for conceiving PCRtechnology.
  • 5. Basic Principle • PCR involves the Invitro primer mediated enzymatic Amplification of the DNA. • Polymerase chain reaction (PCR) is a technology used for quick and easy amplifying DNA sequences, which is based on the principleof enzymatic replicationof the nucleic acids. • Its purpose is to amplify a lot of double-stranded DNAmolecules (fragments) with same (identical) size and sequence by enzymatic method and cyclingconditions.
  • 6. Components of PCR • DNA template (the sample DNA that contains the target sequence to amplify) • Deoxyribonucleoside triphosphates (dNTPs) • PCR buffer • Primers (forward and reverse) • Taq polymerase
  • 7. Steps of polymerase chain reaction-PCR • To perform PCR, extracted sample (target DNA template) is added to a tube containing primers, free nucleotides (dNTPs), and Taq polymerase. • The PCR mixture is placed in a PCR machine. • PCR machine increases and decreases the temperature of the PCR mixture in automatic, programmed steps which generates copies of the target sequence exponentially.
  • 8. PCR STEPS • Initial Denaturation: – Performed only once at the start of PCR – Usually at 95°C for 3-5 min. – Ensures melting of genomic double-stranded DNA. • • Cycling Reactions – Denaturation • usually at 94°C for 30-45 sec • double strand melts (opens) to single stranded DNA. • All enzymatic reactions stop. – Annealing • Most variable step: 54-62°C for 20-45 sec • Primers that fit exactly on double-stranded template DNA bind tighter. – Extension • Taq DNA polymerase optimal temperature: 70-72°C. • Taq DNA polymerase adds dNTP in the 5'→3' direction. – • Final Extension – Varies from 5-10 min. – All single-stranded DNA is double-stranded.
  • 9. This is how PCR works...
  • 10. PCR Cycle - Step 1 Target Sequence Target Sequence Denaturation by Heat
  • 11. PCR Cycle - Step 2 Primers Anneal to Ends of Target
  • 12. PCR Cycle - Step 3 Taq DNA Polymerase Catalyses primer extension as the nucleotides are added. Primer Extension as Nucleotides are Added
  • 13. End of the 1st PCR Cycle Two Copies of Target Sequence
  • 14. Target Amplification No. of No. Amplicon Cycles Copies of 1 2 2 4 3 8 4 16 5 32 6 64 20 1,048,576 30 1,073741824 1 cycle = 2 Amplicon 2 cycle = 4 Amplicon 3 cycle = 8 Amplicon 4 cycle = 16 Amplicon 5 cycle = 32 Amplicon 6 cycle = 64 Amplicon 7 cycle = 128 Amplicon
  • 15. Detection of PCR products • Labeled probe that is specific for the target gene sequence is used to detect PCR amplified gene product (also known as amplicon). Based on the nature of the reporter molecule used, probe generates radioactive, colorimetric, fluorometric, or chemiluminescent signals. Probe based detection of amplicons serves two purposes:
  • 16. • It allows visualization of the PCR product • It provides specificity by ensuring that the amplicon is the target sequence of interest and not the result of non-specific amplification. • Apart from DNA based hybridization method, sometimes simple gel electrophoresis method is sufficient to confirm the presence of specific amplicons.
  • 17. Applications of PCR • Identification and characterization of infectious agents • Direct detection of microorganisms in patient specimens • Detection of antimicrobial resistance • Investigation of strain relatedness of pathogen of interest • Genetic fingerprinting (forensic application/paternity testing) • Detection of mutation ( investigation of genetic diseases) • Cloning genes • PCR sequencing
  • 18. Sources of errors • Mispriming: Primer binds to off target sequence. • Secondary DNA structure can obstruct primer recognition. • Primer dimerize and Amplify (Primer Dimer formation). • Disadventages of PCR include the problems with the post PCR contamination due to the high senstivity (false positive problem except for RT- PCR).
  • 19. conclusion • PCR is not only vital in the clinical laboratory by amplifying small amounts of DNA for STD detection, but it is also important for genetic predisposing for defects such asFactor V Leiden. • The PCR technology can also be employed in law enforcement, genetic testing of animal stocks and vegetable hybrids, and drug screening along with many more areas.