Polymersae chain reaction

1. POLYMERASE CHAIN REACTION
(PCR)
Dr. Jageshwar Mandal Choupal
DNB Resident (pathology)
XEROX
DNA
THERMOCYCLER

2. • Meaning of PCR- Polymerase + Chain reaction.
• It is technique that used to make several copies of small
fragement of DNA and RNA.
• Polymerase-Enzyme that make polymer of any other
molecules , it may be DNA or RNA.
• Chain reaction-A type of chemical reaction which
progresses in exponential manner i.e 1→2, 2→4, 4→8,
8→16 ……
• So within few hundred reactions we can produce billions
of copies of DNA and RNA from single fragement of DNA.

3. • ? Why we want to make to make billions of
copies of DNA
• Most important points are …
• Diagnosis of infectious diseases
• Crime investigation
• Genetic engineering
• Molecular biology.

4. HISTORY OF PCR
• Great mind behind PCR
• Developed PCR in 1985 and awarded noble prize in1993
• PCR machine is otherwise called Thermocycler.
• 1983-Karry banks mulls ,a scientist of Cetus corporation was driving
along US Route 101 in North California,wgen he came up with idea for the
polymerase chain reaction.
• 1985-The pcr was introduced to the scientific community at the
conference in october.Cetus had rewarded kary mullis with a 💰10000
bonus for his invention
• Later,during a corporate reorganisation,Cetus sold the patent for pcr
process to the Pharma company Hoffmnan-LaRoche for 💰300 million.

5. CONTENTS
• PRINCIPAL
• INSTRUMENTS
• REAGENTS
• PRIMER DESIGNING
• MECHENISM OF PCR
• PCR TYPES
• APPLICATIONS
• DISADVANTAGES.

6. PRINCIPLE
• Pcr consists of a series of 20-40 cycles.
• One Pcr cycle comprise of 3 steps
• Denaturation
• Annealing
• Extension
• Temperature used and the length of time applied in each
cycle depends on melting temperature of Dna
polymerase,dNTP,divalent ion concentration.

7. INSTRUMENTS
• Inside it there are 48 to 96 wells are present.
• In which small tubes containg reagents ,small
fragement DNA or RNA ,Taq polymerase,dNTP,
buffer ,mineral oil are kept and inserted in wells
for PCR cyclic reaction.
THERMALCYCLER

8. REAGENTS
• DNA Template
• Primers
• Taq polymerase
• dNTP
• Buffer solution-EDTA,Mgcl2,kcl,Tri-HCL,Triton
x-100,Foemamide.
• Mineral oil or Parrafin beed.

9. DNA TEMPLATE
• PCR reaction need small fragement of DNA and
RNA
• It can be obtained from any source - naso and
oropharyngeal swab.
• Blood
• Hair,Nail
• Tissue obtained from crime scene.

10. • DNA TEMPLATE

11. PRIMER
• Primer is an oligonucleotide sequence- 18 to 26 bp in length
provide free 3”OH for the attatchment of nucleotide bases by
polymerase
• Primers need to match the beginning and the end of the Dna
fragement to be amplified.
• In PCR, both the strands will be amplified.So, one primer each
for both strands must be designed.
• Forward Primer-Beginning of gene of intrest i.e 3”→5” primer
• Reverse Primer-Beginning of complementary strand 5”→3”
PRIMER.
DNA Primer

12. • Primer Uniquness-Knowledge of target sequence bp is important to
design primer
• Primer length-Optimal length of PCR is 18-26bp.
• Long enough for adequate specificity and short enough for primers
to bind easily to the template.
• Primer melting temperature-Temperature at which one half of the
DNA duples will dissociate to become single stranded.
• Primers with melting temperature inthe range of 52 - 56℃ produce
the best results.
• GC contents of the sequence gives fair indication of the primer Tm.

13. • Annealing Temperature- It relies directly on
length and composition of the primers.
• Ta must be set below melting temperature of
Dna primers.
• High Ta -insuffient primer-template hybridisation
• Low Ta- Non -specific binding.
• GC Content-GC content of the primer should be
40 to 55%

14. • GC clamp-
• Presence of G or C bases within last 5 bases of
primers(GC Clamp) promoter specific binding.
• More than 3G” OR C” should be avoided in the
last 5 bases at the 3” end of the primer.
• Primers with long runs of a single bases should
be avoided.
• Maximum no. of runs accepted is 4bp.

15. • Avoid cross homology-To improve specificity of the primers ,it is
necessary to avoid regions of homology.
• Primers designed for a sequence must not amplify other genes in the
mixture.
• Primers are designed and then used in pcr cyclic reaction.
• Repeats-Dinucleotides occuring many times should be avoided.
• Maximum no. of di-nucleotide repeats acceptable in an
oligonucleotide is 4 bp.
• Avoid haipins in primers-Terminal site basepair must be take at most
consideration if we design primer.
• Avoid primer -primer interaction-without it primer cannot bind and
cannot function.

16. Type of DNA POLYMERASES
• 1.Taq polymerases-Thermophilus aquaticus
• Optimum temperature-72 to 75℃
• Half life- 40 min. At 95℃
• Lacks 3”-5” proofreading activity.
• 1 in every 104 nucleotides incorporated.

17. • 2.Pfu dna polymerase
• Isolated from pyrococcus fusiosus
• 3”-5”and 5”-3”exonuclease activity.
• Fidelity of enzyme is 12 fold higher
• Half life at 95℃ -2 hrs.

18. • 3.Pwo DNA polymerase
• 4.Accuzyme DNA polymerase-High fidelity than
Taq polymerase
• 5.KOD Hifi DNA polymerase-High pricessivity and
fidelity
• 6.Tth DNA polymersae-Reverse transcriptase
activity
• 7.M-MLV Reverse Transcriptase-Molony murine
leukemia virus reverse transcriptase

19. MECHENISM OF PCR

20. Details of mech of pcr

21. Initialization step
• 1.-Usually required for Dna polymerases that are
to be activated PCR cycle.
• 2.-Heating around the tenperature 0f 94-96℃ for
10 min.
• 3.-Heating dissociates the inhibitor DNA
polymerase complex.

22. DENATURATION
• 1.-First cyclic event
• 2.-Heating reaction mixture at 94-96℃ for
30sec.
• 3.DNA melted by disrupting the H-bond
between bases yields single stranded DNA.

23. ANNEALING
• 1.-Reaction temperature is lowered to 52 to 55℃for
45 sec.
• 2.-Both forward primer and reverse primer anneals to
the terminal region of complementary bases of target
fragement DNA.
• 3.-High temperature- primer cannot bind to target bp.
• 4.-Annealing temperature should be below the
melting temperature of theprimers used.

24. EXTENSION
• 1.-Temperature depends on polymerase used.
• 2.-Taq polymerase has its optimum activity temperature at
72 - 75℃.
• 3.-DNA polymerase synthesizes new strand complementary
to the DNA template strand by adding dNTPs.
• 4.-Extension time depends on DNA polymerase used as
well as length of the DNA fragement to amplify.
• At its optimum temperature ,DNA polymerase polymerizes
thousands base pair per minute.

25. EXPONENTIAL AMPLIFICATION
Amplification.

26. TEMP. VARIATION GRAPH IN MECHENISM OF
PCR

27. Types of PCR
• 1.Real time PCR
• 2.Reverse transcriptase PCR
• 3.Hot start PCR
• 4.Nested PCR
• 5.Multiplex PCR
• 6.Inverse PCR
• 7.Colony PCR

28. • 8.In situ PCR
• 9.Asymmetric PCR
• 10.Allele specific PCR
• 11.Long PCR
• 12.Long accurate PCR.
• 13. Assembly PCR

29. Real time PCR
• In Real time pcr , process of amplification of DNA is
monitored in real time.
• PCR with an added probe or dye is used to generate
a fluorescent signal from newly formed copy of DNA.
• It is performed in specialized thermal cyclers with
fluorescent detection systems.
• PCR signal is observed as an exponential curve with a
lag phase ,log phase ,linear phase and stationary
phase.

30. • Detection system
• 1.Non-specific detection-SYBr green
dye,BOXTO,Eva Green.
• 2.Specific detection-Taqman probe,molecular
beacon,light up probes and hybridisation
probes.
• 3.Primer based detection-Scorpion
primer,Qzyme and Lux primers.

31. Molecular Beacon assay
• Hairpin shaped molecules with an internally quenched
fluorophore.
• 25 nucleotide long -middls 15 nucleotides are complementary to
target nucleotide and 5 nucleotide at each terminus are
complementary to each other.
• 5” end- fluorophore and 3” end -quencher
• Nucleic acid to be detected is complementary to the strandin the
loop.
• Hybridisation occurs and relieves quenching effect on fluophore.

33. REVERSE TRANSCRIPTASE PCR
• It is used for fragement contaning SSRNA.
• Detect gene expression through the synthesis of complementary DNA (
cDNA ) using reverse transcriptase enzyme.
• Primer used-Random primer,,gene specific primers and oligo dt primer
with 12- 18 bp.
• 1st step- with help of reverse transcriptase cDNA is formed from
SSRNA.
• 2nd step-cDNA is used to form multiple copies by the repeated pcr
cycle.
• Amplified DNA fragement can be analyzed by agarose gel
electrophoresis.

34. NESTED PCR
• Prevents non-specific binding of primer and its amplification
• 2 sets of primers ,used in 2 successive runs of pcr cycle.
• 1st primer binds to the region far away from target
sequence and product is formed.
• Products of 1st primer are used in 2nd PCR reaction with
second set of primers whose 3”end is complementary to
target sequence.
• 2nd PCR has little contamination from unwanted products of
primers ,hairpins, and alternative primer target sequence.

35. INVERSE PCR
• Method used to allow PCR when only one
internal sequence is known.
• Specially useful in identifying flanking sequence
to various genomic inserts.
• This involves series of DNA digestion and self
ligation ,resulting in known sequence at either
end of the unknown sequence.

36. COLONY PCR
• Screening of bacteria(E.COLI) or YEAST for correct ligation or
plasmid products.
• Indiviual transformants can either be lysed in water with a
short heating step or added directly to the pcr reaction and
lysed during the initial heating step.
• Initial heating step causes the release of the plasmid from the
cell,so it can serve as template for the amplification reaction
• Primers designed to specially target the insert DNA can be
used to determine if the construct contains the DNA
fragement of intrest and also insert orientation.

37. APPLICATIOS OF PCR.
• 1.Molecular identification
• Molecular archeology
• Molecular ecology
• Molecular epidemiology
• DNA fingerprinting
• Genetic matching
• Pre- natal diagnosis
• Detection of viral, bacterial n fungal pathogens.

38. • 2.Bioinformatics
• 3.Genomic clonning
• 4.Gene expression studies.

39. ADVANTAGES
• 1. Rapid and easy to perform
• 2.Amplification of DNA from minute sample is
possible.
• 3.Robust,Making it possible to amplify DNA from
degraded samples i.e minute and dead
samples.

40. DISADVANTAGES
• Prior sequence knowledge of target DNA strand
is important.
• Short size range of amplification of products
• Very long target sequence is also not fit for
primer(>1000bp).
• Chances of contamination.

41. THANK YOU