in vitro version of DNA replication......

1. PCR
VIPIN MOHAN
POLYMERASE CHAIN REACTION
VIPIN MOHAN
2011-09-112
College of Agriculture
Vellayani, TVM
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2. The Polymerase Chain
Reaction (PCR) is a
technique to amplify a
piece of DNA very rapidly
outside of a cell
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3. PCR was invented in the 1984 as a way
to make numerous copies of DNA
fragments in the laboratory
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4.  Making copies of short DNA segments.
 DNA Template determines the sequence of
nucleotides.
 Its applications are vast and PCR is now an
integral part of Molecular Biology molecules!!!
 PCR is an in vitro version of DNA amplification.
 Exponential amplification.
PCR
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5. Exponential amplification
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6. The following components are needed to
perform PCR in the laboratory:
1) DNA
2) A heat-stable DNA Polymerase
3) All four nucleotide
triphosphates
4) Buffers(mg++)
5) Primers
6) Thin walled tubes
7) Thermal cycler 6

7. PCR Reagents
 1X Buffer
 10mM Tris-HCl, 50mM KCl
 MgCl2
 1mM - 4mM (1.5mM)
 dNTPs
 200μM
 Primers
 100nM-1μM, 200nm (or less) for real time analysis
 DNA polymerase
 Taq DNA polymerase is thermostable
 1-4 Units (1 unit)
 DNA
 10pg-1μg (20ng) 7

8. DNA synthesizing mixtures
PCR Thermocycler
 Template
 Primers
 dNTP’s
 Taq Polymerase
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9. Template
 DNA molecule which will be amplified.
 Determins the sequence of nucleotide.
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10. Primers
 2 primers must be present to intiate DNA synthesis in
opposite directions from complementary strands.
 A primer is short Nucleic acid that binds to the DNA
template by complementary base pairing .
 Has 3’-OH groups that act as target for the DNA
polymerase.
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11. dNTP’s
 Free nucleotides used as building blocks during
the DNA synthesis.
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12. Taq Polymerase
 DNA polymerase,Cuts the bond between the alpha
& beta phosphate groups.
 Energy acquired from cutting the bonds is used to
produce a new bond.
 Taq polymerase can’t start synthesis on nacked
template.
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13. A cycle of PCR consists of three steps
 DNA denaturation at 95
degrees C.
 Primer annealing at 50-60
degrees C.
 DNA polymerization by a
thermostable DNA polymerase
at 72 degrees C.
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14. 1.Denaturation of DNA
This occurs at 95 ºC mimicking the function of
helicase in the cell.
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15. 2.Annealing or Primers Binding
 Temperature is lowered so primer may
bind to the DNA template.
 There are many copies of primers present
so, primer will prevent the renaturation of
DNA strands.
Forward Primer
Reverse Primer
55^0 c
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16. 3.Extension or Primer Extension
 Polymerase continues the complementary base pairing;
 The 2 DNA double strands will be identical.
 Synthesis will continue as long as template is present.
extension
extension
72^0 C
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17.  The next cycle will begin by denaturing the new DNA
strands formed in the previous cycle
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19. Roles of PCR Reagents
 Taq polymerase
 Enzyme that extends growing DNA strand
complementary to DNA template
 MgCl2
 Provides ions needed for enzyme reaction
 dNTP’s
Nucleotides (Adenine, Cytosine, Guanine,
Thymine) building blocks for new DNA strands
 Buffer
 Maintains optimal pH for enzyme
 Green loading dye
 Adds color and viscosity for future gel loading
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20. Variants of PCR
Hot start e PCR
Touch Down PCR RT- PCR
Asymmetric PCR Long PCR
Nested RACE
Multiplex AP/RAPD
Real time PCR Inverse PCR
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21. Long PCR: Used to amplify DNA over the entire length up to 25kb of genomic DNA
segments cloned.
Quantitative PCR: Product amplification w r t time, which is compared with a standard DNA.
Hot start PCR: Reaction is held at 1000C for few minutes.
Asymmetric PCR:Generally used for sequencing,Yields single stranded
product( For enrichment )
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22. Multiplex PCR
 Use of multiple primers to amplify different gene
regionsHelpful in detecting gene deletions.
 Multiplex-PCR consists of multiple primer sets within
a single PCR mixture to produce amplicons of varying sizes
that are specific to different DNA sequences.
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23. Nested PCR
 Involves two consecutive PCR reactions of 25 cycles.
 The first PCR uses primers external to the sequence of interest.
 The second PCR uses the product of the first PCR in conjunction
with one or more nested primers to amplify the sequence within
the region flanked by the initial set of primers.
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24. RT PCR
 Gene expression studies
 Analysis of RNA sequences
 Diagnosis of infectious agents
 Diagnosis of genetic diseases
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25. Random Amplification of cDNA ends
(RACE)
 Used to obtain the full length sequence of an RNA
transcript
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26. Touch Down PCR
 Annealing Temperature is reduced by 1degree or so and
continued till 10-15 cycles
 To enrich the target sequences
 94,65,72- 94, 64,72, 94,63,72--------------94,50,72( 20
cycles)
 The anealing temperature during a polymerase chain
reaction determines the specificity of primer annealing.
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27. AP/RAPD
 Uses single primer of arbitrary sequence for PCR
and amplifies random segments of genomic DNA
 Primers are small in size, usually decamers.
 Does not require any specific knowledge of the
DNA sequence of the target organism
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28. Inverse PCR
Used to amplify DNA of unknown sequence that is adjacent to known DNA sequence.
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29. e-PCR
 Electronic PCR refers to a computational procedure that is
used to search DNA sequences for sequence tagged sites
(STSs), each of which is defined by a pair of primer
sequences and an expected PCR product size.
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30. Q-PCR/ Real Time PCR
 The amplified DNA is detected as the reaction progresses
in real time.
 By using a fluorescent reporter in the reaction, it is
possible to measure DNA generation.
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31. Real Time PCR
 Real-time PCR analysis detects specific nucleic acid amplification products as they
accumulate in real-time.
Reporter Dye- FAM , TET Quencher- TAMRA
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32. Some applications of PCR
 Forensic medicine.
 Preimplantation Genetic Diagnosis (PGD).
 Archeology.
 Paternity testing.
 Identify species
 Identify alleles/genotypes to assess variability in a
population
 Create sequences for phylogenies to determine taxonomic
relationships
 Conduct forensic investigations
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33.  PCR is NOT used to:
 Amplify RNA or proteins
 Construct genomic or cDNA libraries
 Make monoclonal antibodies
 Conduct stem cell research
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35. Thank you
THANK YOU
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