it gives the idea about different types of polymerise chain reaction

1. SHEHANA.K. SALIM

2. Introduction
 Making more copies of DNA from a single copy of DNA
is called gene amplification.
 PCR is a technique of gene amplification.
 The in vitro amplification of DNA by repeated cycles of
strand separation and polymerization by DNA
polymerize activity, is called polymerase chain reaction
(PCR).
 In each polymerization , the number of DNA
fragments gets doubled .
 It makes millions or billions of copies from a single
DNA within a short time,i.e. less than 5hrs.

3.  The PCR was invented by Kary Banks Mullis in 1985 and
was awarded nobel prize in 1993.

4. Thermal cycler
PCR amplification mix typically
containing
 DNA Template
 Primers
 Taq polymerase
 Deoxynucleoside
triphosphates(dNTPs)
 Buffer solution
 Divalent cations(eg.Mg2+ )
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Requirements of PCR

5. How does it work?
PCR is carried out in a single tube kept in a thermal
cycler which is programmable to set alternate heating
and cooling.

6. 3 stages
I. Heat denaturation
 The reaction mixture is heated at 94⁰C for 1 min. This
temp. denatures the double stranded DNA into two
individual strands.
II. Primer annealing
 In the presence of an excess dNTPs(the building blocks of
new DNA material), oligonucleotide primers are added.
The primers are complementary to either end of the target
sequence but lie on opposite strands. As the mixture cools
slowly at a lower temp. (54⁰C) for 1.5 mints. During this
time, one primer binds with 3’ end of one DNA strand and
the other primer binds with the 3’ of its complementary
strand. This process is called primer annealing.

7. III.Primer extension
 72⁰C temp.is provided to the reaction mixture for 1 min. As
this temp. is suitable for Taq DNA polymerase,it adds
complementary nucleotides one by one to the 3’- OH
group of the primer.
 As a result, the second DNA strand grows to the full size.
This process is called polymerase activity.
 At the end of the 1st cycle ,two identical double stranded
DNAs are formed from a DNA in the reaction mixture.
Each cycle completes within 5 mints.
 Each of the two duplex DNAs formed in the first cycle
enters into the second cycle. The cycle is repeated several
times.
 The basic reactions of PCR are the same in all cycles. In
each cycle, the number of duplex DNAs gets doubled ,i.e.
2-4-8-16-32-64-128.
 Total time for 1 cycle= approx 4 mints

8. Amplification

9.  Following PCR ,the amplification product can be
detected using gel electrophoresis where visualization
of a band containing DNA fragments of a particular
size can be indicating the presence of the target
sequence in the original starter DNA sample.
Similarly ,absence of a band may indicate that the
target sequence was not present in the original starter
DNA sample.
PCR can be used in combination with other techniques
to not just simply amplify DNA but also to detect
specific target sequence.

10. Use PCR to amplify gene fragment
a. template DNA is melted (94C)
3’ 5’
5’ 3’
3’ 5’
5’ 3’
b. primers anneal to complementary site in melted DNA (55C)
3’ 5’
5’ 3’
3’ 5’
5’ 3’
c. two copies of the template DNA made (72C)

11. Denaturation: DNA melts
Annealing: Primers bind
Extension: DNA is replicated
PCR Animation

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NEW AUTOMATED PCR
OLD PCR

13. Variants of PCR
PCR is highly versatile technique and has been
modified in variety of way to suit specific
applications.
Single sided PCR
Inverse PCR
Anchored PCR
Random PCR
RT-PCR
Long accurate PCR (LA-PCR)
Quantitative Real time PCR (Q-RT PCR)
Allele specific PCR

14. Single sided PCR
 It is a slightly variant of conventional PCR.
 Here only one type of oligonucleotide primer is
included in the reaction mixture.
 The primer anneals with 3’end of the DNA strand
which is complementary to the DNA strand to be
amplified.
 The other strand does not involve in the
polymerisation.
 Taq polymerase adds complementary nucleotides to
the primer and synthesizes a full DNA strand.Thus a
duplex DNA is formed.

15.  The new strand never acts as template and it gets
accumulated in the reaction mixture.
 The parent strand is used as template for several times
to amplify complementary strand.
USES
Single sided PCR is used to propagate single stranded
DNAs to make hybridisation probes.
Such probes can be used in the clinical diagnosis of
genetic diseases and pathogenic diseases and in
detecting genes (DNAs) in a sample.

16. Inverse PCR
 In this method,the flank sequences that lie behind the
primer binding sites in the DNA are amplified.
 It is the inversion of the conventional PCR in which
flanking sequence have not been amplified.Here, the
DNA fragment is rearranged before the start of PCR.
 The DNA fragment to be amplified is cut out from the
source DNA using a restriction enzyme and then the cut
ends are linked by DNA ligase. As a result, a circular
duplex DNA is formed.
 Two primers are designed in such a way as to anneal with
the core sequence lying between the flank regions.

17.  The primers and circular DNAs are added in to the
PCR reaction mixture.
 At 95˚C the DNA strands separate into circular single
stranded DNAs.
 At 55˚C the two primers attach with the respective
strand and polymerization takes place at 72˚C .
 Because of its reaction , two circular DNAs are formed.
There is a nick in the daughter strand.

18.  When the reaction mixture is heated again to 95˚C,the
two DNAs release their daughter strands in the form of
linear DNAs.
 At 55˚C the two daughter strands hybridise with each
other to form a linear duplex DNA with flanking
regions as single stranded extensions.
USE
Inverse PCR is used in chromosome walking.

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20. Anchored PCR
- A small sequence of nucleotides can be
attached or tagged to target DNA.
- The anchor is frequently a poly G to
which a poly C primer is used.
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22. Random PCR
 A synthetic DNA strand containing two or three
internal primers is designed and amplified in the first
PCR.synthetic DNA with two or more internal primers
is called nested primer.
 In the second PCR the synthetic DNA binds on the
DNA strand to be amplified and then polymerization
proceeds.
 If one or two internal primers failed anneal with the
target DNA stand, polymerisation does not take place.
So the sought-afterDNA clone is selectively amplified
in this method.

23. uses
Random PCR is used to propagate a specific DNA
strand in a pool of DNA fragments without amplifying
the other strand
It is used in RAPD(Random Amplified Polymorphic
DNA ) analysis.

24. RT-PCR
 This PCR refers to reverse transcription followed by
polymerase chain reaction.
 Here reverse transcriptase and a primer are added to
the conventional PCR reaction mixture.
 The reverse transcriptase synthezises cDNA strand to
form a RNA-DNA hybrid .
 The second DNA strand is synthesized on the c DNA
strand by Taq polymerase.As a result cDNA clones are
formed.

25.  If the mRNA is primed with oligo(d)T primer, cDNA
are made from different kinds of mRNAs in the
reaction mixture. Such cDNA clones are used to make
c DNA library.
 If the primer is nested one, it selectively forms cDNAs
from a particular mRNA and amplifies the cDNA
clones. This will help for rapid screening.

27. Rapid Amplification of cDNA
Ends(RACE)
 In this PCR some sequence at 3’end or 5’ end of the
mRNA is made into cDNA and the latter is made to
synthesize the second strand .
 The duplex cDNA clone so formed is amplified by
PCR.
 Oligo(d)T primer with 5’extension and a primer
designed so as to anneal at the middle of the cDNA
strand are used to amplify the cDNA ends.

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 -Used to obtain 3' and 5' end sequence of
cDNA transcripts.

29. uses
RACE is used to amplify one end of cDNA cloned upto
106 copies
Such selectively amplified sequences are useful for the
aminoacid sequence analysis

30. Long accurate PCR (LA-PCR)
 Amplification of long DNA fragments is desirable for
numerous applications of gene manipulation .The
basic PCR works well when small fragments are
amplified
 The efficiency of amplification and therefore the yield
of amplified fragments decrease significantly as the
size of the amplicon increases over 5kb.
 This decrease in the yield of longer amplified
fragments is attributable to partial synthesis across the
desired sequence ,which is not a suitable substrate for
the subsequent cycles.

31.  Barnes and cheng et al. examined the factors affecting
the thermostable polymerisation across larger regions
of DNA and identified key variables affecting the yield
of longer PCR fragments.
 Most significant of these was the absence of a 3’-5’
exonuclease (proof reading) activity in Taq
polymerase.
 When the Taq polymerase misincorporates a dNTP,
subsequent extention of the strand either proceeds
very slowly or stops completely.
 To overcome this problem,a second thermostable
polymerase with proof reading capability is added.

32. Quantitative Real Time PCR
 This PCR enables us to quantify the amount of starting
material(target sequence).
 Higuchi et al pioneered the use of ethidium bromide
to quantify PCR products as they accumulate.
 Amplification produces increasing amounts of double
stranded DNA, which binds ethidium bromide,
resulting in an increase in fluorescence .
 By plotting the increases in fluorescence versus cycle
no. It is possible to analyse PCR kinetics in real time.

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34.  The principal drawback to the use of ethidium bromide is
that both specific and non-specific products generate a
signal.
 This can be overcome by the use of probe based methods
for assying product accumulation.
 The probes used are oligonucleotides with a reporter
fluorescent dye attached at the 5’ end and a quencher dye
at the 3’ end.While the probe is intact,the proximity of the
quencher reduces the fluorescence emitted the reproter
dye.
 If the target sequence is present the probe is anneal
downstream from one of the primer sites.As the primer is
extended, the probe is cleaved by the 5’nuclease activity of
the Taq polymerase.This cleavage of the probe separate the
reporter dye from the quencher dye.Thereby increasing the
reporter-dye signal

35.  Cleavage removes the probe from the target strand
allowing primer extension to continue to the end of
the template strand.
 Additional reporter-dye molecules are cleaved from
their respective probe with each cycle, effecting an
increase in fluorescence intensity proportional to the
amount of amplicon produced.
 Instrumentation has been developed which combines
thermal cycling with measurement of fluorescence,
thereby enabling the progress of the PCR to be
monitored in real time.

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Allele- Specific PCR
• Selective PCR amplification of the alleles to detect
single nucleotide polymorphism (SNP).
• Selective amplification is usually achieved by
designing a primer such that the primer will match or
mismatch one of the alleles at the 3’ end of the primer.

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Other types of PCR
 Overlap extension PCR
 Assemble PCR
 Helicase dependent amplification PCR
 Intersequence-specific PCR(ISSR)
 Ligation-mediated PCR
 Methylation –specific PCR
 Miniprimer PCR
 Multiplex PCR
 Nested PCR
 Solid phase PCR
 Touch down PCR and so on…………………..

39. Applications of PCR

40.  PCR is used to propagate DNA for gene manipulation
and for constructing DNA libraries.
 It helps in the clinical diagnosis of genetic
&pathogenic disease.
 It can be used to determine the sex of the baby at
embryo level.
 It is used to detect the criminals in forensic science.
 Single sided PCR is used to make gene probes to detect
genetic diseases, pathogenic diseases and to detect the
presence of a particular gene in the sample.

41.  Inverse PCR is used in chromosome walking to detect the
correct order of various genes in a chromosome.
 Random PCR is used to amplify a particular DNA in a pool
of DNAs.
 RT-PCR cDNA copies selectively from a single species
mRNA found in a pool of mRNAs.
 RACE amplifies a particular portion of cDNA.
 PCR is used to add restriction sites or promoter or part of
another gene to the one end of amplified DNA by the
incorporation of 5’extra sequence.

42.  The PCR can be used to detect the presence of a gene
transferred into an organism(transgene) by using the end
sequences of the transgene for amplification of DNA from
the transgenic organism. Amplification will occur only
when the transgene is present in the oraganism; the
amplified DNA is detected as a band on the
electrophoretic gel.
 Microdissected segments of chromosomes, e.g., of salivary
gland chromosomes of Drosophila can be used for PCR
amplification to determine the physical location of genes
in chromosomes.

43.  Sequence Information
Amplicon size
 Error rate during amplification
Sensitivity to inhibitors
Contamination
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