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8/3/2019 DNA amplification by PCR
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DNA amplification by
PCR
Y. Vijay Surya.
KVSR SCOPS
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Polymerase Chain Reaction
Invented by Kary B Mullis
(Given Noble Prize in 1993)
• It is a fast, inexpensive and cell free method of DNA
cloning.
• At the end of this method we get the multiple copies of
targeted DNA sequence
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Method : Three steps
1.Denaturation
2.Annealing
3.Extension
Requirements :
•DNA that contains sequence of interest •Primers OR Oligonucleotides ( 2 in No.)
•DNA Polymerase
•Mixture of four deoxynucleoidtriphosphate
Additives :
•PCR buffer, mgcl2, DMSO, formamide,
glycerol, etc..
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STEP 1: Denaturation
• Heat the reaction mixture at 94 ºc
• So, double stranded DNA molecule
converts in to single stranded DNA
• ssDNA acts as a template for the
primers and DNA polymerase
STEP 2: Annealing
• Reaction mixture is cooled at 50-60 ºc
• It allows the primers to anneal with the
templates ( two single strands )
STEP 3: Extension
• Temperature is again raised to 72 ºc
• So, Taq Polymerase adds new nucleotides
and synthesize complimentary strands
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Primers :
• Two in numbers
• Complimentary to the targeted DNAsequence at 3’end
• 15-20 bp long
• 10-100 picomol of primers required for
100-1000 bp of target sequence
Targeted sequence :
• 100-5,000 bp long (Long PCR allows
42 kbp to be amplified 10-20-10-15 or
1- 105 DNA copies per 100 µl )
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Taq Polymerase :
• Thermo stable polymerase
• Obtained from “Thermus Aquatiqus”
• Optimum temperature – 72 ºc• Can be stable up to 94 ºc
Some other Thermo stable polymerases
• Thermus thermophilus • Thermotoga martima
• Thermococcus litoralis
• Pyrococcus furiosus
After Step-3 temperature is increased up
to 94º c again for denaturation for next cycle
After completion of first cycle next 20-35
cycle can perform.
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Cell Based cloning
Very tedious-it may take weeks
Costly compared to PCR
Not sensitive than PCR
Separation of individual DNA clone by comparing
with genomic DNA library can be done
Robustness is not there – amplification can’t done
from material in which DNA
is badly degraded or embedded
Sequence of targeted DNA need not to be known
Proof-reading is possible
Cloning by pcr
Easy and speedy – may take 3-5 min
Very economic,
-Unsophisticated instrument is used
Very sensitive
Minute amount of DNA can be cloned
-Even from single cell
Not done
Robustness-
Amplification can done from material in which
DNA is badly degraded or embedded
Sequence of targeted DNA must be known
Proof-reading is not possible
(vent polymerase can be used although not fully
efficent )
Sort sized limited amount of product obtained at
last
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Instrument : Thermal cycler
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Applications :
•In forensic laboratory in DNA testing
•To study DNA polymorphisum•In molecular mapping
•In prenatal diagnosis
•In DNA fingerprinting / In DNA typing
•In detection of pathogen and disease based on DNA•For detection of bacterial and viral infection
•For monitoring cancer therapy
•In PCR based diagnostic tests like AIDS, Lyme disease,
Hepatitis etc..
•For detecting mutations
•In RNA amplification by RT-PCR
•In DNA labeling
•In sexing the embryos
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Real-Time PCR:
• While traditional PCR uses agarosegels for detection of PCR amplification
at the final phase of end-point of the
PCR reaction,
• it allow for the detection of PCRamplification during the early phases of
the reaction.
RT-PCR:
Reverse-Transcriptase PCR
For amplification of RNA
RNADNAamplification
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Acknowledgement
N.KANAKA DURGA DEVIAsst. Professor
KVSR Siddhartha college of pharmaceutical
sciences,Vijayawada-520010