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Technique of Polymerase Chain
 Reaction (PCR)- Experimental
 Biotechnology



Presented by
Dr. B. Victor., Ph.D.,
email : bonfiliusvictor@gmail.com
blog : bonvictor.blogspot.com
Presentation out line

      • Polymerase chain reaction (PCR)
        (Enzymatic Amplification of DNA)
      • Origin and definition of PCR
      • PCR –a DNA copying machine
      • Requirements of PCR
      • Thermostable DNA polymerases
      • Critical steps in PCR
      • Step cycle programme
      • PCR protocol
      • Variants of PCR
      • Characterization of PCR product
      • Problems and limitations
      • Merits and applications
Polymerase chain reaction (PCR)
(Enzymatic Amplification of DNA)


        PCR is a novel molecular
        technique involving in
        vitro enzymatic
        replication of defined
        DNA sequences.
Definitions of PCR
Definition-1
• A technique for amplifying DNA sequences in
  vitro by separating the DNA into two strands
  and incubating it with oligonucleotide
  primers and DNA polymerase.


Definition -2
• A biochemical technique used in Molecular
  Biology to amplify a specific fragment of
  target DNA.
Origin of Polymerase chain reaction
(PCR)
        In vitro DNA synthesis
        • PCR was discovered by Kary B. Mulis in
          1983 of Cetus Corporation, a Biotech
          company in California, USA.
        • He won the Nobel Prize for Chemistry in
          1993 for ‘contributions to the developments
          of methods within DNA-based chemistry’.
        • ‘Taq polymerase’ an enzyme used in PCR was
          described as ‘molecule of the year’ 1989.
A copying machine for DNA molecules


         PCR multiplies a single, microscopic
         strand of the DNA molecule into
         billions of times within hours.
         PCR had a major impact on
         recombinant DNA technology.
         PCR has multiple applications in
         medicine, genetics, biotechnology, and
         forensics.
PCR-A DNA multiplication technology
PCR is a powerful technique, in which from a
single copy of a DNA molecule, millions of copies
can be obtained with high accuracy, specificity and
in a very short time.


     DNA amplification process in PCR is cyclical and
     the concentration of DNA doubles at each cycle.



           The total amount of DNA concentration
           increases exponentially during the cyclical
           process of PCR machine.
The ‘master mix’ components
for PCR machine
     1. A thermostable DNA polymerase:
        tag polymerase
     2. A template DNA
     3. A complete set of deoxynucleotide
        triphosphates e.g. dATP, dCTP,
        dGTP and dTTP
     4. Tris buffer of pH 8.8
     5. A pair of oligonucleotide primers
     6. Mg 2+ and detergents
     7. 2-mercaptoethanol to stabilize
        proteins during thermal cycle.
Requirements for PCR
DNA template – DNA segment to be amplified.
Two primers- a short segment of DNA ( forward and
 reverse primers) about 20–25 bases long .
Taq polymerase – an enzyme to synthesize DNA copies.
Deoxynucleotide triphosphates – the building blocks for
 new DNA strand.
Buffer solution – a suitable chemical environment.
Divalent cations – Mg 2+ ions
Monovalent ions – Potassium ions
PCR machine – a thermal cycler
Thermostable DNA polymerase
• The thermophilic DNA polymerases catalyze
  template-directed synthesis of DNA from
  nucleotide triphosphates.
• Several thermostable polymerase enzymes are
  used in PCR
Pfu DNA polymerase- Pyrococcus furiosus
Vent polymerase- Thermococcus litoralis
Taq polymerase- Thermus aquaticus
Oligonucleotide primers
• They are synthesized chemically to be
  complementary to sequences which flank the
  region of DNA to be amplified.
• They are usually about 20-25 nucleotides in
  length.
• The primers are designed to anneal specifically
  to the opposite strands of the template molecule.
• It is the specificity of the primer annealing
  reaction which ensures that the PCR amplifies
  the appropriate region of the template DNA.
Critical steps in PCR




  Sample       Target      Primer
Preparation   selection   selection
3 – temperature cycle in PCR

Temperature - 90-980C - separates two
strands of target DNA.


    Temperature – 40-600C anneals two
    complementary primers to the ends of
    separated single strands of target DNA

        Temperature 720 C allows taq
        polymerase to use ss target DNA and
        primers to synthesize new strands.
DNA thermal cycler use
          ‘Step cycle’ programme


                            Denature     Anneal at
                             at 940C     550C for
                            For 20 sec    20 sec



                                Extend at 720C
                                  for 30 sec

*For a total of 30 cycles                   *Overall single cycle time is 3.75 min.
Sources of sample material for PCR




                     Drop of
 Hospital           dried from     Cells of   Sperm or   Mouth wash
  tissue    hair   blood from     mummified    sperm
specimens          the scene of    material    lysates      Etc.
                      crime
PCR procedure-cycle of amplification
            Denaturation
              reaction




      Extension      Annealing
       reaction       reaction
PCR protocol
1.   Denaturation of ds DNA template –melting target DNA-it is
     the thermal denaturation of the dsDNA at 950C for 1 min.
2.   Annealing of two oligonucleotide primers – 680C for 60 sec.
     The annealing temperature is dependent on the length and
     G+C content of the primer sequences.
3.   Polymerase extension of dsDNA molecules – temp. raised
     at 750C for about 30 sec.
     The step cycle programme makes the instrument to heat
     and cool to the set temperatures due to solid state Peltier-
     effect device , which actively modulates the desired
     temperature. There may be as many as 30-35 cycles.
PCR protocol
PCR-DNA synthesis cycle
Laboratory PCR technique
             Prepare the Master Mix of reagents
                    and aliquot into tubes




                    Add DNA template(s)




           Program thermal cycler, load with tubes
                        and start




              Remove tubes and analyze results
Variants of PCR
1. Standard PCR – sequences of both ends of target
   DNA have to be known. Two primers define the
   ends of target DNA and only that part is amplified.
2. Single sided PCR – Here DNA is rearranged before
   amplification so that only one primer is needed.
   This is also called Anchored PCR.
3. Inverse PCR – DNA at primer sites rather than
   between two primers is amplified because primer
   sites which are bracketing may have important
   sequence like promoter for triggering target gene
   into action.
Characterization of PCR product
• Contamination of the reagents by foreign DNA
  or annealing of primers to alternative sites in the
  template DNA may produce unwanted DNA
  molecules.
• Gel electrophoresis – to assess purity of product.
• Multiple bands in the electropherogram suggest
  primers annealing to multiple sites.
• Smear of DNA suggests presence of excess
  template DNA.
Uses of PCR
                     Cloning




Detection                               Forensic
of ancient                                DNA
   DNA                                  detection
                      Uses
                     of PCR



         Detection             Identifying
          of viral             transgenic
         infection               plants
Problems and limitations

 Contamination of reaction       PCR can not substitute for
mixture by bacteria, viruses,     cell- based gene cloning ,
and our own DNA presents a      when large amounts of a gene
       real problem .                    are desired.


 Taq polymerase used in
  PCR often lack 3' to 5'       PCRs of longer products are
exonuclease activity. This      less efficient due to enzyme
enzyme lacks the ability to     activity loss. Applies only to
 correct mis-incorporated          short DNA fragments
       nucleotides.
Merits of PCR

              Simplicity

              Specificity

             Much faster

 Generate and modify DNA fragments of
     defined length and sequence
Applications of PCR -1
 Detection of pathogens in food, water and
  tissue specimens.
 Detection of tuberculosis, AIDS and other
  microbial diseases.
 Diagnosis of genetic diseases-e.g. sickle cell
  anemia, β-thalasemia, hemophilia
 Identification of criminals, disputed
  parentage.
 Monitor gene expression in genetic
  engineering or gene therapy experiments.
Applications of PCR -2
To study genetic profile of animals and to trace
 evolutionary and cultural lineage of human
 beings.
To study DNA polymorphism.
To determine orientation and location of
 restriction fragments relative to one another.
To conduct microbial surveillance of the
 environment.
Summary - applications of PCR

  PCR is used in medical and biological
  research, including cloning, genetic analysis,
  genetic fingerprinting, diagnostics, pathogen
  detection and genetic fingerprinting


  PCR is valuable to scientists in gene
  mapping, the study of gene functions and cell
  identification.
References
• Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and
  Erlich, H. (1985). Enzymatic amplification of beta-globin
  genomic sequences and restriction site analysis for
  diagnosis of sickle cell anemia. Science 230: 1350-54
• Mullis, K. and Faloona, F. (1987). Specific synthesis of
  DNA in vitro via a polymerase-catalyzed chain
  reaction. Methods Enzymol 155: 335-350.
• Mullis, K. (1990). The unusual origin of the polymerase
  chain reaction. Scientific American April 56-65
• Rabinow, P. (1996). Making PCR: A story of
  biotechnology. University of Chicago Press
   Dr.B.Victor is a highly experienced professor,
    recently retired from the reputed educational
    institution- St. Xavier’ s College, Palayamkottai,
    India-627001.
   He was the dean of sciences, IQAC coordinator
    and assistant controller of examinations.
   He has more than 32 years of teaching and
    research experience
   He has taught a diversity of college courses and
    guided 12 Ph.D scholars.
    Send your comments to :
    bonfiliusvictor@gmail.com
Technique of polymerase chain reaction (pcr) experimental biotechnology

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Technique of polymerase chain reaction (pcr) experimental biotechnology

  • 1. Technique of Polymerase Chain Reaction (PCR)- Experimental Biotechnology Presented by Dr. B. Victor., Ph.D., email : bonfiliusvictor@gmail.com blog : bonvictor.blogspot.com
  • 2. Presentation out line • Polymerase chain reaction (PCR) (Enzymatic Amplification of DNA) • Origin and definition of PCR • PCR –a DNA copying machine • Requirements of PCR • Thermostable DNA polymerases • Critical steps in PCR • Step cycle programme • PCR protocol • Variants of PCR • Characterization of PCR product • Problems and limitations • Merits and applications
  • 3. Polymerase chain reaction (PCR) (Enzymatic Amplification of DNA) PCR is a novel molecular technique involving in vitro enzymatic replication of defined DNA sequences.
  • 4. Definitions of PCR Definition-1 • A technique for amplifying DNA sequences in vitro by separating the DNA into two strands and incubating it with oligonucleotide primers and DNA polymerase. Definition -2 • A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA.
  • 5. Origin of Polymerase chain reaction (PCR) In vitro DNA synthesis • PCR was discovered by Kary B. Mulis in 1983 of Cetus Corporation, a Biotech company in California, USA. • He won the Nobel Prize for Chemistry in 1993 for ‘contributions to the developments of methods within DNA-based chemistry’. • ‘Taq polymerase’ an enzyme used in PCR was described as ‘molecule of the year’ 1989.
  • 6. A copying machine for DNA molecules PCR multiplies a single, microscopic strand of the DNA molecule into billions of times within hours. PCR had a major impact on recombinant DNA technology. PCR has multiple applications in medicine, genetics, biotechnology, and forensics.
  • 7. PCR-A DNA multiplication technology PCR is a powerful technique, in which from a single copy of a DNA molecule, millions of copies can be obtained with high accuracy, specificity and in a very short time. DNA amplification process in PCR is cyclical and the concentration of DNA doubles at each cycle. The total amount of DNA concentration increases exponentially during the cyclical process of PCR machine.
  • 8. The ‘master mix’ components for PCR machine 1. A thermostable DNA polymerase: tag polymerase 2. A template DNA 3. A complete set of deoxynucleotide triphosphates e.g. dATP, dCTP, dGTP and dTTP 4. Tris buffer of pH 8.8 5. A pair of oligonucleotide primers 6. Mg 2+ and detergents 7. 2-mercaptoethanol to stabilize proteins during thermal cycle.
  • 9. Requirements for PCR DNA template – DNA segment to be amplified. Two primers- a short segment of DNA ( forward and reverse primers) about 20–25 bases long . Taq polymerase – an enzyme to synthesize DNA copies. Deoxynucleotide triphosphates – the building blocks for new DNA strand. Buffer solution – a suitable chemical environment. Divalent cations – Mg 2+ ions Monovalent ions – Potassium ions PCR machine – a thermal cycler
  • 10. Thermostable DNA polymerase • The thermophilic DNA polymerases catalyze template-directed synthesis of DNA from nucleotide triphosphates. • Several thermostable polymerase enzymes are used in PCR Pfu DNA polymerase- Pyrococcus furiosus Vent polymerase- Thermococcus litoralis Taq polymerase- Thermus aquaticus
  • 11. Oligonucleotide primers • They are synthesized chemically to be complementary to sequences which flank the region of DNA to be amplified. • They are usually about 20-25 nucleotides in length. • The primers are designed to anneal specifically to the opposite strands of the template molecule. • It is the specificity of the primer annealing reaction which ensures that the PCR amplifies the appropriate region of the template DNA.
  • 12. Critical steps in PCR Sample Target Primer Preparation selection selection
  • 13. 3 – temperature cycle in PCR Temperature - 90-980C - separates two strands of target DNA. Temperature – 40-600C anneals two complementary primers to the ends of separated single strands of target DNA Temperature 720 C allows taq polymerase to use ss target DNA and primers to synthesize new strands.
  • 14. DNA thermal cycler use ‘Step cycle’ programme Denature Anneal at at 940C 550C for For 20 sec 20 sec Extend at 720C for 30 sec *For a total of 30 cycles *Overall single cycle time is 3.75 min.
  • 15. Sources of sample material for PCR Drop of Hospital dried from Cells of Sperm or Mouth wash tissue hair blood from mummified sperm specimens the scene of material lysates Etc. crime
  • 16. PCR procedure-cycle of amplification Denaturation reaction Extension Annealing reaction reaction
  • 17. PCR protocol 1. Denaturation of ds DNA template –melting target DNA-it is the thermal denaturation of the dsDNA at 950C for 1 min. 2. Annealing of two oligonucleotide primers – 680C for 60 sec. The annealing temperature is dependent on the length and G+C content of the primer sequences. 3. Polymerase extension of dsDNA molecules – temp. raised at 750C for about 30 sec. The step cycle programme makes the instrument to heat and cool to the set temperatures due to solid state Peltier- effect device , which actively modulates the desired temperature. There may be as many as 30-35 cycles.
  • 20. Laboratory PCR technique Prepare the Master Mix of reagents and aliquot into tubes Add DNA template(s) Program thermal cycler, load with tubes and start Remove tubes and analyze results
  • 21. Variants of PCR 1. Standard PCR – sequences of both ends of target DNA have to be known. Two primers define the ends of target DNA and only that part is amplified. 2. Single sided PCR – Here DNA is rearranged before amplification so that only one primer is needed. This is also called Anchored PCR. 3. Inverse PCR – DNA at primer sites rather than between two primers is amplified because primer sites which are bracketing may have important sequence like promoter for triggering target gene into action.
  • 22. Characterization of PCR product • Contamination of the reagents by foreign DNA or annealing of primers to alternative sites in the template DNA may produce unwanted DNA molecules. • Gel electrophoresis – to assess purity of product. • Multiple bands in the electropherogram suggest primers annealing to multiple sites. • Smear of DNA suggests presence of excess template DNA.
  • 23. Uses of PCR Cloning Detection Forensic of ancient DNA DNA detection Uses of PCR Detection Identifying of viral transgenic infection plants
  • 24. Problems and limitations Contamination of reaction PCR can not substitute for mixture by bacteria, viruses, cell- based gene cloning , and our own DNA presents a when large amounts of a gene real problem . are desired. Taq polymerase used in PCR often lack 3' to 5' PCRs of longer products are exonuclease activity. This less efficient due to enzyme enzyme lacks the ability to activity loss. Applies only to correct mis-incorporated short DNA fragments nucleotides.
  • 25. Merits of PCR Simplicity Specificity Much faster Generate and modify DNA fragments of defined length and sequence
  • 26. Applications of PCR -1  Detection of pathogens in food, water and tissue specimens.  Detection of tuberculosis, AIDS and other microbial diseases.  Diagnosis of genetic diseases-e.g. sickle cell anemia, β-thalasemia, hemophilia  Identification of criminals, disputed parentage.  Monitor gene expression in genetic engineering or gene therapy experiments.
  • 27. Applications of PCR -2 To study genetic profile of animals and to trace evolutionary and cultural lineage of human beings. To study DNA polymorphism. To determine orientation and location of restriction fragments relative to one another. To conduct microbial surveillance of the environment.
  • 28. Summary - applications of PCR PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting PCR is valuable to scientists in gene mapping, the study of gene functions and cell identification.
  • 29. References • Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and Erlich, H. (1985). Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350-54 • Mullis, K. and Faloona, F. (1987). Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155: 335-350. • Mullis, K. (1990). The unusual origin of the polymerase chain reaction. Scientific American April 56-65 • Rabinow, P. (1996). Making PCR: A story of biotechnology. University of Chicago Press
  • 30. Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier’ s College, Palayamkottai, India-627001.  He was the dean of sciences, IQAC coordinator and assistant controller of examinations.  He has more than 32 years of teaching and research experience  He has taught a diversity of college courses and guided 12 Ph.D scholars.  Send your comments to : bonfiliusvictor@gmail.com