In this slide briefly describe some important note on pcr,rapd,and aflp,which helps to understand the students about this normally . I wish for your future goal that you will shine one day inshallah . Thank you for watching

1. A Presentation on
PCR, RAPD & AFLP
Md. Nahidul Islam
Dept. of Genetic Engineering and Biotechnology.
Jessore University of Science and Technology.
Jessore 7408,Bangladesh

2. Outline
 PCR
• History
• Definition
• Requirement or Reaction
components
• Procedure of PCR
• Example of PCR programme
• Benefits of PCR
• Limitations of PCR
• Application of PCR

3.  RAPD
 Principle
 Definition
 Modification of RAPD
 Procedure of RAPD
 Advantages
 Drawbacks
 Application

4. AFLP
 Introduction
 Definition
 Procedure of AFLP
 Benefits
 Drawbacks
 Application

5. PCR
Polymerase Chain Reaction
History
• Invented by Kary Mullis 1983
• 1985: First publication of PCR by Cetus
Corporation appears in Science.
• 1986: Purified Taq polymerase is first used in
PCR
• 1993:Dr. Kary Mullis Received Nobel Prize in
Chemistry for conceiving PCR technology.

6. Definition
An in-vitro amplification technique allows
synthesizing millions of copies of the gene or
DNA of interest from a single copy.
Also called “polymerase” because the only
enzyme used in this reaction is DNA
polymerase

7. Requirement or Reaction components
• Template (DNA/RNA/Plasmid)
• Oligonucleotide Primer
• Deoxynucleotide triphosphates or dNTPs
(Mixture of dATP, dTTP, dCTP, dGTP)
• Taq DNA Polymerase
• Buffer system
• MgCl2
• Water

8. Procedure of PCR
• A PCR cycle consist of 3 steps :
1.Denaturation:
• Reaction mixture heated to 94°C for 1 minute.
• The temperature denatures dsDNA into two
single strands due to breakage in weak
hydrogen bonds.

9. 2.Primer annealing:
• The reaction temperature is rapidly lowered to
54-60°C for 1.5 minute.
• The primers to bind (anneal) to their
complementary sequence in the template
DNA.
3.Elongation:
• Also known at extension, this step usually
occurs at 72°C

10. • Addition of nucleotides by Taq polymerase.
• Duration to complete cycle 4-5 min.
• In each cycle the number of DNA gets doubled-2-
4-8-16……
• A reaction usually consists of 30-35 cycle forming
about million copies

11. Fig: PCR procedure

12. Example of PCR programme
• Initial denaturation 94°C for 5 mins
• Thermo-cycle file - 30 cycles of
• Denaturation : 94°C for 1 mins
• Annealing : 55°C -30 secs to 1mins
• Extension : 72°C for 45 secs
• Final extension 72°C for 5 mins
• Holding ( soak ) fileusually 4°c

13. Benefits of PCR
• Specific amplification.
• Rapid.
• Most accurate and feasible technique.
• Extremely sensitive.
• Easy of use.

14. Limitations of PCR
• Setting up and running requires high technical
skill.
• High equipment cost.
• High test cost.
• High sterile environment should be provide

15. Application of PCR
• To amplify DNA fragments isolated from
organisms.
• Used to detect genetic disease.
• In forensic science , in DNA fingerprinting
• PCR products can be used as probes
• Pre-natal diagnosis
• Gene therapy

16. RAPD
Principle
Abbreviation for Random Amplified
Polymorphic DNA.
 A PCR based molecular marker technique.
 Invented by Williams et al. (1990).
 Needs one primer for amplification.

17. Definition
A molecular technique in which random DNA are
amplified by the polymerase chain reaction
using single primers of arbitrary nucleotide
sequence.
It is a type of PCR reaction, but the segments of
DNA that are amplified are random.

18. Modification Of RAPD
 AP-PCR (Arbitrarily Primed PCR)
 DAMD (Directed Amplification of
Mini-satellite Region DNA).
ISSR(Inter-Simple Sequence Repeat).

19. AP-PCR
• Arbitrary Primed PCR
• Amplification occurs in three parts
• Higher concentration primer is used at first
cycle
• Analyzed on acryl amide gels
• Detected by autoradiograph

20. DAMD
• Directed Amplification of Minisatellite Region
DNA
• Use VNTR as primer
• Detect polymorphism

21. ISSR
• Inter-Simple Sequence Repeat
• Used as Molecular Marker
• Lying between adjacent Microsatellites
• Used to detect polymorphism

22. Protocol of RAPD analysis
Taq DNA polymerase, Primer, dNTPs
and Buffer
DNA isolation
Keep the tubes in PCR
thermo cycler
DNA strand separated
Annealing of primer
(36ºC, 2min)
Denature DNA
(94°C, 1min)

23. Primer annealed of template DNA
strands
DNA synthesis
(72ºC, 1.5min)
Complementary strand
synthesis
Amplified products separated by
gel electrophoresis
35 to 45 cycle

24. Fig: RAPD procedure

25. Advantages of RAPD
• Quick and efficient screening for DNA
• Involves no radioactive assays.
• Requires a small amount of DNA.
• Requires no probes.
• Involves no blotting or hybridization steps.

26. Drawbacks of RAPD
• Efficiency and low expensive.
• Low reproducibility.
• Highly sensitive and complicated procedure.
• Markers are dominant.
• PCR cycling conditions greatly influence the
out come.

27. Application of RAPD
• Gene mapping.
• DNA fingerprinting.
• Phylogenetic analysis.
• Mapping of traits.
• Identification of the somatic hybrids.
• Analysis of genetic structure

28. AFLP
Introduction
• Full form is ‘Amplified Fragment Length
Polymorphism.
• A PCR based method; developed by Vos et al.
in 1995.
• A combination of RFLP and RAPD methods.
• Detect polymorphism.

29. Definition
A technique used to amplify genomic
restriction fragments from a complex mixture
of DNA fragments which is generated by
specific restriction endonucleases.
Technique in which differences in restriction
fragments are revealed by PCR.

30. Procedure of AFLP
Following steps are involved in AFLP process
 Restriction Digestion
 Adaptor ligation
 Amplification
 Electrophoresis

31. Restriction Digestion
Restriction endonucleases are used in
digestion.
 Generally two RE is used.
- One is 4 base cutter (MseI)
- Other is 6 base cutter (EcoRI)
MesI 5’TTAA3’
EcoRI 5’GAATTC3’

32. Adaptor Ligation
 Two end specific adaptors (a different one for each
enzyme) are ligated to the digested fragments.
 In case of MesI and EcoRI
-one adaptor will complement to the Msel cut end
-the other will complement to the EcoRI cut end
DNA with sticky
end
Adaptor 1 Adaptor 2
Adaptor 2Adaptor 1

33. Amplification
• PCR is used
• Amplification occurs for selected fragments
only
• Two PCR primers complementary to the two
adaptors
• Primers are labeled with radioactive or
fluorescence dye

34. Electrophoresis
 Polyacrylamide gel is used for separating DNA
bands.
30-100 DNA bands can be detected
 Selected fragments will show in band

35. Fig: Procedure of AFLP

36. Benefits of AFLP
Used for genome mapping.
 Highly polymorphic.
 High reproducibility.
 Different parts of the genome can be
analyzed.
 Discriminates heterozygote's from
homozygote's by using gel scanner.

37. Drawbacks of AFLP
Complex procedure.
 Technically more demanding than RAPD.
 Requires experience of sequencing gels.
Highly expensive and requires more DNA than
RAPD

38. Application Of AFLP
Creation of genetic maps for new species
Determination of relatedness among cultivars
Establishment of linkage groups in crosses
Useful in genetic studies such as biodiversity
evaluation, genotyping etc.
 In criminal identification and paternity tests.