Types of PCR

1. M.V.MALA
I- Msc BIOTECHNOLOGY
DEPARTMENT BIOTECHNOLOGY

2. INDEX
• INTRODUCTION
• HISTORY
• TYPES OF PCR
 MULTIPLX
 NESTED
 REVERSE TRANSCRIPTASE
 REAL TIME
 TOUCHDOWN
 HOT START
 COLONY
• CONCLUTION

3. INTRODUCTION
Polymerase chain reaction (PCR) is technique that result
in exponential amplification of a desired region of a DNA
molecule in vitro.
With technique, small amount the genetic material can be
amplified (ie, to make a huge number of copies of DNA).
HISTORY:
 The of PCR technique was invented by KARY MULLIS.
 The work mullis received the 1993 NOBLE PRIZE in
chemistry.

4. WHY POLYMERASE
 Because the only enzyme used in the reaction is DNA
polymerase.
WHY CHAIN
 Because the products of the first reaction become the
substrates of the following one and so on.

5. TYPES OF PCR
 Multiplex
 Nested
 Reverse transcriptase
 Real time
 Touchdown
 Hot start
 Colony

6. MULTIPLEX
 Multiplex PCR is a variant of PCR. which enabling
stimultaneous amplification of many targets of interest
in one reaction by using more than one pair primer.
 It is a special type of PCR used for detection of multiple
pathogen by using multiple primer sets each one targets
a particular pathogens.

7. TYPES OF MULTIPLEX
 single template PCR
reaction
 Multiple template PCR
reaction
ADVANTAGE
 Internal controls
 Efficiency
 Indication of template of
quality

8. APPLICATION
 Pathogen Identification
 High trough put SNP mutation analysis genotypin
 Gene deletion analysis
 Template quantification
 Linkage analysis
 RNA detection
USES:
This permits the stimultaneous analysis of multiple target
in a single sample

9. NESTED
 Nested PCR is a modified from of PCR designed to
reduce the comtamination in products occurs due to
non-specific amplification (or) non-specific primer binding
sites.
 Nested PCR makes use of two sets of amplification
primer.
 The target DNA sequence for one set of primer ( termed
'INNER' primer ) in situated within the target sequence
for the second set of primer ( termed 'OUTER' primer ).

10.  In practice 'OUTER' primer are first used in a standard PCR
procedure for a test sample. then 'INNER' primer are used in
second PCR reaction.
 The product generated first PCR serves amplification target
for two PCR reaction.
 In procedure Sensitivity of assay increased to multiple
fold as product of one reaction re-amplified in the two
reaction.
 Specificity assay improved as inner primer will only amplify
specific product obtained in one PCR reaction.

11. ADVANTAGES
 Very low probability
of non-specific
amplification.
USES
 Detection of pathogen
that occur with very
few amount
.

12. REVERSE TRANSCRIPTASE
 Based on the process of reverse transcriptase, which
reverse transcriptase RNA into DNA and initially
isolated from retroviruses.
 First step of RT-PCR first strand reaction synthesis of
cDNA using oligo dt primer (37`C) 1hr.
 Second strand reaction digestion of cDNA : RNA hybrid
(RNase H) standard PCR with DNA oligo primer.
 Allow the detection of even rare or low copy mRNA
sequence by amplifying its complementary DNA.

13. ADVANTAGE
Useful in a
generating large
cDNA libraries from
small amounts of
mRNA.

14. APPLICATION
 Detection of RNA virus like (HIV)
 Detection of infections agents
 Diagnosis of genetic mutation
 Gene expression in a tissue
RACE-PCR ( Rapid amplification of cDNA end )
used to obtain 3' - 6' end sequences of cDNA transcripts

15. COLONY
 Colony PCR is used for the screening recombinants from
bacterial, bacteriophage (or) yeast transformation products.
eg: screen for correct DNA vector construction
SIGNIFICANCE
Colony PCR is a fast and reliable method for the screening
of recombinants.
 Biotechnology products

17. TOUCHDOWN
 Touchdown PCR offers a simple and rapid means to
optimize PCR specificity, sensitivity and yield, instead
of lengthy optimization and primer redesigning.
 TD-PCR was an initial annealing temperature above the
estimated melting temperature (Tm) of the primer in
use. the progressively moves down to lower , more
tolerent annealing temperature as the PCR cycle
continues successively.

18.  Annealing T` early cycle usually 3-5` C above Tm
primer used and later cycles is similar amount below the Tm.
 Any difference in melting T` between correct and incorrect
annealing will result in a exponential two fold advantage per
cycle.
 TD-PCR has special application for amplification o T` are
usually difficult to amplify.
 It can also be used as a standard to enhance specificity
and product.

19. REAL TIME
 Real time polymerase chain
reaction is a laboratory technique
of molecular biology based on the
polymerase chain reaction.
 It monitors the amplification of a
target DNA molecular during the
PCR.

20. PRINCIPLE
 Fluorescence Resonance Energy Transfer [FRET]
some commonly used flurophores for labeling probes.
 Improving fluorescence signal detection.
TECHNIQUE
 Non-specific detection using DNA binding dyes.
 Specific detection target specific probes.

21. ADVANTAGE
 Qualification of gene expression
 Genotyping
 Methylation detection
 DNA damage measurement
 Viral quantification

22. HOT START
 This is a technique that reduce non-specific aplification
during the initial set up stages of the PCR.
 The technique may be performed manually by heating
the reaction components to the melting temperature
(eg: 95`C) before adding the polymerase.
 DNA polymerase :
Eubacterial type I DNA polymerase

23.  This method involved, with holding one of critical
components of PCR like:
Megnesium
The enzymes DNA polymerase
PCR primers
dNTPs from the reaction until
the temperature in the first cycle rise above the annealing
temperature.

24. CONCLUTION
 Numerous types of PCR exist and the basis on which
they exist vary: ranging from this application areas,
specificity, amplification target etc.
 These all combined gives us an array of impassibles in
the exploitation of genes in various fields of life.

25. THANK YOU