The document is a presentation on asymmetric PCR, detailing its principles, applications, and variations in the context of polymerase chain reaction techniques. Asymmetric PCR is described as a method that preferentially amplifies one strand of DNA for applications like sequencing and hybridization probing. The presentation also discusses the basic requirements for PCR, advantages, and limitations associated with the technique.

2. A
Presentation
on
Asymmetric PCR

3. Presented by…..
Md. Afzalur Rahman
Asraful Islam Rayhan
Md. Shaharul Islam
Shamsun Nahar Lipa
Dept. of Pharmacy
Jessore University Of Science & Technology..

4. Contents
 What is PCR
 Basic Requirements
 Types of PCR
 Asymmetric PCR
 Applications of PCR
 Advantages of PCR
 Limitations of PCR

5. Two methods currently exist for
amplifying the DNA or making copies
 Cloning—takes a long time for enough
clones to reach maturity
 PCR—works on even a single molecule
quickly

6. PCR: stands for Polymerase Chain Reaction
A technique that allows the production of million of
copies of a single Target DNA fragment.
PCR is an in vitro technique that uses a few basic
everyday molecular biology reagents to make large
numbers of copies of a specific DNA fragment or a
specific region of a DNA strand in a test-tube.

7. The nature of the primers allow the addition of any sequence or molecule to
the 5’ end of the primer.
The sequence of the borders of the PCR product is the sequence
of the primers not the sequence of the target DNA

8. The nature of the primers allow the addition of any sequence or molecule to
the 5’ end of the primer.
During PCR, heating the DNA will separate the 2 strands
Then specific primers anneal to complementary sequence
DNA polymerase attaches to the 3’ side of the primer and constructs
the complementary strand

9. The nature of the primers allow the addition of any sequence or molecule to
the 5’ end of the primer.
DNA polymerase attaches to the 3’ side of the primer and constructs
the complementary strand

10. The nature of the primers allow the addition of any sequence or molecule to
the 5’ end of the primer.
If we have a non complementary part on the 5’ side,
the reaction can still proceed

11. The nature of the primers allow the addition of any sequence or molecule to
the 5’ end of the primer.
But if we have a non complementary part on the 3’ side
the reaction CAN NOT proceed

12. Primers resemble a key

13. You can attach to the primer many functional sequences
like restriction sites

14.  DNA Template
 Primers
 Taq polymerase
 Deoxynucleoside
triphosphates(dNTPs)
 Buffer solution
 Divalent cations(eg.Mg2+
)
Requirements of
PCR

15. Variations of PCR...
 Nested PCR
 Inverse PCR
 Anchored PCR
 Reverse transcription PCR (RT-PCR)
 Asymmetric PCR
 Real-time quantitative PCR
 Random amplified polymorphic DNA (RAPD)
 Amplified fragment length polymorphism (AFLP)
 Rapid amplification of cDNA ends (RACE)

16. What
is
Asymmetric PCR

17.  Asymmetric PCR is used to
preferentially amplify one strand of the
original DNA more than the other.
 It finds use in some types of sequencing
and hybridization probing where having
only one of the two complementary
stands is ideal.
 PCR is carried out as usual, but with a
great excess of one primers for the
chosen strand.

18. Asymmetric PCR: Synthesis of single strand DNA

19. Asymmetric PCR: Synthesis of single strand DNA

20. Asymmetric PCR: Synthesis of single strand DNA
In a regular PCR the same amount of forward and reverse primers is
added

21. Asymmetric PCR: Synthesis of single strand DNA
In an asymmetric PCR the one of the primers is largely in excess
compared to the other

22. Asymmetric PCR: Synthesis of single strand DNA

23. Gene cloning: Using restriction enzymes
Gene 1 Gene 2 Gene 3
PCR
Ligation

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

25. Advantages:
PCR IN CLINICAL DIAGNOSIS
PCR IN DNA SEQUENCING
PCR IN FORSENIC MEDICINE
PCR IN GENE MANIPULATION AND EXPRESSION
STUDIES
PCR IN COMPARATIVE STUDY OF GENOMICS
PCR IN COMPARISON WITH GENE CLONING

26. Limitations
SEQUENCE INFORMATION
AMPLICON SIZE
 ERROR RATE DURING AMPLIFICATION
SENSITIVITY TO INHIBITORS
CONTAMINATION
ARTEFACTS

27. 27