principle, history , advantage, and disadvantage of Pcr

1. PRESENTED BY :- RAJAT SINGH
MSC FOOD TECH
PCR

2. Topics to be covered today
1. Introduction
2. Brief History
3. PCR KINETICS
4. PCR Principles
5. PCR Requirements
6 How can I detect PCR products?
7.Advantages & disadvantages of PCR
8.PCR applications

3. Introduction
• PCR (Polymerase chain reaction) is an in vitro technique for
the amplification of a specific sequence of DNA..
• The reaction depends mainly on the activity of DNA
polymerase which stand on 3’ end of annealed primers and
start building the complementary strands.
• PCR reaction involves a number of repettitive cycles
composed of three steps ( denaturation ,annealing and
extension). These cycles result in the amplification of the
target gene.

4. History
• The invention of PCR emerged from researches conducted in
the early 1980s at cetus corporation in California.
• In 1985 Karry Mullis developed PCR and was awarded
nobel prize in1993

5. History contd..
…Early , there are two obstacles facing
the researchers ;
1) The klenow fragment of DNA polymerase 1 extracted from
E.coli that used in PCR reaction was not thermostable and
inactivated by denaturing temperature 95 “C used to separate
the double – stranded DNA at the beginning of each cycle.
2) PCR was laborious ,slow , and required manual transfer of
the reaction tubes between water baths of different temperture.

6. History contd..
1) The first obstacle was overcome by using Taq polymerase
purified from a hemophilic bacterium Thermopiles aquaticus
which resides in hot springs.
2) The second obstacle was overcome by using thermocycler
which invented by cetus and then subjected to a series of
enhancements the thermal cycler we are using now.
• The thermocycler can be preprogrammed to repeat the three
steps usually for between 25 and 40 cycles.

7. PCR Kinetics

9. Principles
• PCR consists of a number of repetitive cycles (25-40).Each
cycle is formed of 3 main steps ;-
1. Denaturation :- The two strand are separated by breaking
down the hydrogen bonds between complementary bases at
92- 96’C. (2minutes)
2 .Annealing :- Two specific primers are linked to sequence
flanking the target region ( 37 -65’C)(1min)
3.Primer Extension :- The heat stable ( eg. Taq polymerase
extends from the 3’ end of both primers to synthesize a
complimentary strand of both parents strand .( 72’C) ( 2min)

11. Requirements of PCR
1. DNA template :- source ; Bacteria , Viruses , plant animal…etc
concentration : (100 ng – 1Ug)
2. DNA Polymerase :- eg taq , Tth, Pfu… etc
 Concentration : 0.5 – 2.5 unit
 High conc.:- Non specific reaction
 Low conc. : Low PCR yield
3.PCR buffer :- standard buffer contains :
 10mM tris HCL, 50mM KCL, 0.5 -2.5 mM MgCl2
 The concentration of MgCl2 should be optimized
 If increase (non specific reaction)
 If decrease ( low PCR yield)

12. Requirement of PCR
4) dNTPs ( A,T,C and G) :- Higher dNTPs concentration
force Taq DNA polymerase to misincorporate deoxy-
ribonucleotides which in turn affects the enzymes fidelity. Also
, higher concentration of dNTPs results in trapping of free
mg2+ which necessary for the enzyme activity. The optional
concentration of dNTPs for Taq polymerase activity is 200 uM
5) PCR Primers :- Concentration : 0.1 – 0.5 uM ( 10-50pml)

13. Criteria should be fulfilled in your primers
;
1. Mostly Two primer are used : forward and reverse primer
2. Length ; 17- 30 nucleotides
3. G-C content ( 40 -60 %)
4. Melting temperature ( 55 – 65’C)
5. Flanking the region to be amplified
6. Average nucleotide distribution

14. How Can I detect the PCR product ?

15. How Can I detect the PCR product
?

18. Advantages of PCR
1) High degree of sensitivity : It detect even a single copy of the target
sequence present in the DNA samples and generates millions copies of
this sequence.
2) Simple :- Semi- automated ( most of work carried out by PCR machine
and ellectrophoresis unit , no complex steps ( just pipetting and mixing
of reagents ) less efforts ( about 60 min. working time)
3) Rapid ( 3-4 hours to get results)
4) Extremely specific :- specific primers produce specific amplicon of
specific size
5) versatile :- used with any micro-organism ( just change the primers)
6) Even degraded DNA i.e unsuitable for gene cloning can be used in
PCR.

19. Disadvantage of PCR
1 Qualitative data :- positive and negative data
2:- Not fully automated
3) Risk of ethidium bromide ( carcinogenic)

20. Application of PCR
1) In the field of scientific research
• For DNA sequencing
• DNA cloning
• Identification of different organism
2) In forensic filed:-
• DNA fingerprinting almost based on PCR.
• Establish paternity and other family relationships.
3) RT-PCR : provides the information of activity of tumor cell
and viruses.
4) Detection of food borne bacteria.

21. Thank you