This document describes polymerase chain reaction (PCR), including its history, principles, components, procedures, applications, and limitations. PCR is a technique used to amplify a specific DNA sequence, allowing millions of copies to be generated. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Key components of PCR include DNA template, primers, DNA polymerase, nucleotides, and buffer solutions.

1. PCR
(POLYMERASE CHAIN
REACTION)
GROUP MEMBERS
HIBA AKHTAR(SP18-BSI-
024)
KASHAF NAZ(SP18-BSI-
025)

2. Table of contents:
 What is PCR?
 Short history of PCR
 Principle of PCR
 Purpose of PCR
 Components of PCR
 Procedure of PCR
 Variants of PCR
 Applications of PCR
 Problems and limitations with PCR
 Conclusion

3. What is PCR?
 PCR stands for polymerase chain reaction.
 It is the technique widely used in molecular
biology to make several copies of a specific
DNA segment
 PCR is a technique that takes specific
sequences of DNA of small amount and
amplifies it to be used in further testing.
 Makes millions of copies of a particular
segment of DNA in a given time span
 In vitro technique

4. Short history of PCR
 1983: Dr.kary mullis developed PCR
 1985: first publication of PCR by cetus corporation
appears in science.
 1986: purified Taq polymerase was first used in PCR.
 1988: PerkinElmer introduces the automated thermal
cycler.
 1989: science declares Taq polymerase “molecule of
the year”
 1990: amplification and detection of specific DNA
sequence using a fluorescent DNA-binding dye, laying
the foundation for future “real-time” or “kinetic” PCR
 1991: RT-PCR for diagnostic tests for RNA viruses
was discovered using single thermo stable
polymerase.

5. Purpose of PCR
 The purpose of the polymerase chain reaction
is to amplify a lot of double-stranded DNA
molecules (fragments) with same length
having same (identical)sizes and sequences
by using the enzymatic method and the
process is in the form of cyclic conditions

6. Principle of PCR
 PCR involves the primer mediated enzymatic
amplification of DNA . PCR is based on using the
ability of DNA polymerase to synthesize new
strand of DNA complementary to the offered
template strand.
 Primer is needed because DNA polymerase can
add a nucleotide only onto a preexisting 3’-OH
group to add the first nucleotide .
 DNA polymerase then elongate its 3 end by
adding more nucleotides to generate an extended
region of double stranded DNA

7. Components of PCR
The PCR technique requires the following components:
1. DNA template: The double stranded DNA (ds
DNA) of interest , separated from sample.
2. DNA polymerase: usually a thermo stable Taq
polymerase that does not rapidly denature at high
temperature (98 degrees) , and can function at a
temperature optimum of about 70 degree centigrade
3. Oligonucleotide primers: short pieces of single
stranded DNA (often 20-30 base pairs) which are
complementary to 3’ ends of the sense and the
antisense strands of the target sequence.

8. 4. Deoxynucleotide triphosphate : single
units of bases A, T,C,G (d ATP , d TTP , d GTP
,d CTP) provide the energy for polymerization
and the building blocks for DNA synthesis.
5. Buffer system : includes magnesium
potassium to provide the optimal conditions for
DNA denaturation and renaturation ; also
important for polymerase activity , stability and
fidelity.

9. Procedure of PCR
The process of PCR is start by mixing all the
PCR components together and taken through
the series of 3 major cyclic reactions ,
conducted in an automated , self-contained
thermo cycler machine . The all important
feasible conditions are very crucial to be
provided to carry out the procedure of
polymerase chain reaction efficiently.

10. Denaturation
This step involves heating the reaction mixture
to 94 degree centigrade for the duration of 15-
30 seconds. During this process , the double
stranded DNA is denatured to single strands
due to breakage in weak hydrogen bonds.

11. Denaturatin process

12. Annealing
the reaction temperature is rapidly lowered to
54-60 degree centigrade for the time span of
20-40 seconds not longer of that . This step
allows the primers to bind (anneal) to the
complementary sequence in the template DNA
. So that the procedur of PCR can begin.

13. Annealing process

14. Elongation
This step is also known as extension , this
step usually occurs at 70-80 degree
centigrade (most commonly 72 degree
centigrade) .72 degrees is the optimum
temperature for the Taq polymerase to built the
complementary strand .it attaches to the
primer and then adds DNA bases to the single
strand one-by-one in 5’ to 3’ direction. DNA
polymerase will add about 1,000 bp/minute

15. Elongation process

16. Process of PCR

17. Variants of PCR
1. Standard PCR : sequences both ends of
target DNA have to be known . Two primers
define the ends of target DNA and only that part
is amplified.
2. Single sided PCR : here DNA is rearranged
before amplification so that only one primer is
needed .this is also called Anchored PCR.
3. Inverse PCR : DNA at primer sites rather than
between two primers is amplified because primer
sites which are bracketing may have important
sequence like promoter for triggering target gene
into action.

18. Applications of PCR
 PCR is used in cloning .
 PCR is also being widely used in forensic DNA
detection.
 PCR technique can also be used to identify the
transgenic plants.
 PCR can be used for the detection of viral infections.
 PCR is used in the detection of ancient DNA for the
evolutionary study purposes.
 PCR in comparative study of genomics.
 PCR is also used in gene manipulation and
expression study.
 PCR in DNA sequencing.

19. Problems and limitations with
PCR
 Contamination of the reaction mixture by
bacteria, viruses and our own DNA presents a
real problem.
 PCR can not substitute for a cell-based gene
cloning, when large amounts of gene are
desired.
 Taq polymerase used in PCR often lacks 3’ to
5’ exonuclease activity .this enzyme lacks the
ability to correct mis-incorporated nucleotides.
 PCRs of longer products are less efficient due
to enzyme activity loss . Applies only to short
DNA fragments.

20. conclusion
 PCR is not only vital in clinical laboratories by
amplifying small amounts of DNA for STD
detection but also important for genetic
predisposing for defects such as factor V Leiden.
 The PCR technology can also be employed in law
enforcement ,genetic testing of animal stocks and
vegetable hybrids , and drug screening along with
many more areas.
 PCR is a very important time saving technique to
make a huge number of copies of a specific
sequence of DNA required.