Polymerase chain reaction PCR types APPLICATION OF PCR

1. Polymerase Chain
Reaction
Dr.Kamlesh shah
PSSHDA,KADI

2. Introduction
• PCR, polymerase chain reaction, is an in-vitro
technique for amplification of a region of DNA
whose sequence is known or which lies between two
regions of known sequence
• Before PCR, DNA of interest could only be
amplified by over-expression in cells and this with
limited yield

3. History
1. 1966, Thomas Brock discovers Thermus
Aquaticus, a thermostable bacteria in the hot
springs of Yellowstone National Park
2. 1983, Kary Mullis postulated the concept of PCR (
Nobel Prize in 1993)
3. 1985, Saiki publishes the first application of PCR (
beta-Globin)
4. 1985, Cetus Corp. Scientists isolate Thermostable
Taq Polymerase (from T.aquaticus), which
revolutionized PCR

4. PCR is……..
• Polymerase Chain Reaction
• An in vitro method for the enzymatic synthesis of specific
DNA sequences, using two oligonucleotide primers that
hybridize to opposite strands and flank the region of interest
in the target DNA.
• It’s a means of selectively amplifying a particular segment of
DNA.
• The segment may represent a small part of a large and
complex mixture of DNAs. e.g. a specific exon of a human
gene.
• It can be thought of as a molecular photocopier.

5. Principle…
• An in vitro method for enzymatic synthesis of DNA
• Reaction uses two oligonucleotide primers that hybridize to
opposite strands and flank the region of interest.
• A heat stable DNA polymerase catalyses the elongation of
primers.
• Primers extension products serve as template in next cycle.
• Numbers of target copies double in each cycle.

6. 6
What’s Requirement for PCR?
Genomic DNA
5’ 3’
3’ 5’
primers
A
B Free
nucleotides
Taq DNA
polymerase
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Mg2+
Buffer
containing
magnesium

7. The Basic Protocol--Denaturation
Genomic DNA
95oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’

8. The Basic Protocol--Annealing
~55oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’
5’
Genomic DNA
A
B
primers
A
B

9. The Basic Protocol-Extension
72oC
5’ 3’
3’ 5’
5’ 3’
3’ 5’
5’
5’
Genomic DNA
Taq polymerase

10. Former DNA amplification in vitro
• Water baths- Three different temperature
92-94 º C 55 º C 74 º C
• Tubes were moved physically-manually
• Large volume, More chemical - Costly
• Klenov fragment used to amplify DNA
(DNA polymerase from E. coli)

11. PCR Reaction Condition
Steps Temperature Time
Denaturing 94 o C 20-30 sec
Annealing 55o C 20-60 sec
Extension 72 o C 30-60 sec
72oC
Extending
94oC
Denaturizing
55oC
Annealing

12. 1- DNA template
• DNA containing
region to be
sequenced
• Size of target DNA
to be amplified : up
to 3 Kb

13. 2- Primers
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically produced
• complimentary to the 3’
ends of target DNA
• not complimentary to
each other

14. Primers (ctnd)
• Not containing inverted repeat sequences to avoid
formation of internal structures
• 40-60% GC content preferred for better annealing
• Tm of primers can be calculated to determine
annealing T0
• Tm= .41(%G+C) + 16.6log(J+) + 81.5 where J+ is
the concentration of monovalent ions

15. 3-Enzyme
• Usually Taq Polymerase or anyone of the natural or
Recombinant thermostable polymerases
• Stable at T0 up to 950 C
• High processivity
• Taq Pol has 5’-3’ exo only, no proofreading

21. Standard thermocycle

22. RT-PCR
• Reverse Transcriptase PCR
• Uses RNA as the initial template
• RNA-directed DNA polymerase (rTh)
• Yields ds cDNA

26. Detection of amplification products
• Gel electrophoresis
• Sequencing of amplified fragment
• Southern blot
• etc...

27. 27
• Aims to reduce nonspecific background by gradually lowering the
annealing temperature as PCR cycling progresses.
• The annealing temperature at the initial cycles is usually a few
degrees above the Tm of the primers used, while at the later cycles,
it is a few degrees below the primer Tm
• The higher temperatures give greater specificity for primer
binding, and the lower temperatures permit more efficient
amplification from the specific products formed during the initial
cycles.
• This ensures that only specific annealing of the primers to their
correct target sequence takes place before any non specific
annealing events occur
Touch Down PCR

28. • Two pairs of PCR primers for a single locus in two successive
reactions.
• First reaction- one pair to generate DNA products (consist non-
specifically amplified DNA fragment)
• Second reaction- with a set of primers whose binding sites are
completely within the DNA target fragment
• The second pair of primers (nested primers) bind within the first
PCR product and produce a second PCR product shorter than the
first one
• If the wrong locus were amplified by mistake, the probability is
very low that it would also be amplified a second time by a
second pair of primers.
• Increases the specificity of DNA amplification, by reducing
background due to non-specific amplification of DNA.
Nested PCR

29. 29
Enables simultaneous amplification of many targets of
interest in one reaction
Multiple, unique primer sets within a single PCR reaction to
produce amplicons of varying sizes specific to different
DNA sequences.
By targeting multiple genes at once, additional information
may be gained from a single test run that otherwise would
require several times the reagents and more time to perform.
Annealing temperatures for each of the primer sets must be
optimized to work correctly within a single reaction, and
amplicon sizes, i.e., their base pair length, should be
different enough to form distinct bands when visualized by
gel electrophoresis
Multiplex PCR

30. Applications of PCR
Basic Research Applied Research
• Genetic matching
• Detection of pathogens
• Pre-natal diagnosis
• DNA fingerprinting
• Gene therapy
• Mutation screening
• Drug discovery
• Classification of organisms
• Genotyping
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• Bioinformatics
• Genomic cloning
• Site-directed mutagenesis
• Gene expression studies

31. Applications of PCR
Molecular Identification Sequencing Genetic Engineering
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
• Bioinformatics
• Genomic cloning
• Human Genome Project
• Site-directed mutagenesis
• Gene expression studies

32. MOLECULAR IDENTIFICATION:

34. Detection of Unknown Mutations
Molecular Identification:

35. Classification of Organisms
1) Relating to each other
2) Similarities
3) Differences
* Fossils
* Trace amounts
* Small organisms
! DNA !
Molecular Identification:
Insufficient data

36. Detection Of Pathogens
Molecular Identification:

37. Prenatal Diagnosis
644 bp
440 bp
204 bp
Molecular analysis of a family with an autosomal recessive disease.
Molecular Identification:
• Chorionic Villus
• Amniotic Fluid

38. SEQUENCING
Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP)
NO polymerisation after a dideoxynucleotide!
Fragments of DNA differing only by one nucleotide are
generated
Nucleotides are either
or

39. Applications
• Genome mapping and gene function determination
• Biodiversity studies ( e.g. evolution studies)
• Diagnostics ( prenatal testing of genetic diseases,
early detection of cancer, viral infections...)
• Detection of drug resistance genes
• Forensic (DNA fingerprinting)

40. Advantages
• Automated, fast, reliable (reproducible) results
• Contained :(less chances of contamination)
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols

41. Conclusion
The speed and ease of use, sensitivity, specificity and robustness of
PCR has revolutionised molecular biology and made PCR the most
widely used and powerful technique with great spectrum of
research and diagnostic applications.

42. References
• Fundamentals of Biochem ( Voet, Voet, Pratt)
• Molecular Cell Biology ( Lodish, Darnell..)