This document summarizes techniques for analyzing DNA including PCR, RAPD, and AFLP. It describes PCR as a biological reaction that amplifies DNA using primers, enzymes, and thermal cycling. RAPD uses random primers in PCR to analyze genetic relatedness, while AFLP is a more powerful technique that combines RAPD and RFLP. Both RAPD and AFLP can analyze whole genomes without prior sequence knowledge and produce dominant markers for genetic mapping, fingerprinting, and trait analysis.

1. Jessore University of Science & Technology
a presentation
on
PCR,RAPD & AFLP
Presented by
Md Robel Ahmed
Roll No: 140603
4th Year 1st Semester
Dept. of Genetic Engineering
& Biotechnology
Presented to
Forhad Karim Saikot
Assistant professor
Dept. of Genetic Engineering
& Biotechnology

2. Contents
 PCR(Polymerase Chain Reaction)
 RAPD(Randomly Amplified
Polymorphic DNA)
 AFLP(Amplified Fragment Length

3. Polymerase Chain Reaction
 Biological reaction.
 Simple and ingenious method.
 Kary Mulis in 1985.
 Amplify DNA into millions of copies.
 Utilize low quantity of specimen
 Having numerous applications.

4. PCR requires the following
 DNA template
 Primer(forward and reverse primer)
 Enzymes(taq polymerase)
 (nucleoside triphosphate) dNTPs
 MgCl2
 Water

5. PCR
Denaturation
Extension
Annealing of
primer
Mainly completed in
three steps
Mechanism of PCR

6. Denaturati
on:
• Melting of target
DNA
• 94ºC at 1-2
minute
• Origination of
template strand
3’
3’
3’
3’
5’
5’
5’
5’
Mechanism of PCR

7. Mechanism of PCR
Annealing of primer:
• Temperature 50-70ºC based
on DNA template
• Primers binds to complementary
sequence
• Requires at least 60 seconds
oligonucleotide sequence
• Primer added in excess
• Temperature calculated as
T=2(AT)+4(G+C)
reverse primer
Forward primer
5’
5’
3’
3’
5’
5’ 3’
3’

8. Extension:
• Nucleotide triphosphate & thermostable
DNA polymerase added
• Tem 72ºC at 0.5 to 3 minute
• Taq polymerase binds and
Extends the DNA
5’
5’
5’
5’
5’3’
3’
3’
3’
3’
3’ 5’
5’
5’
Mechanism of PCR

9. Points to be remembered
1. These three steps repeats several times.
2. Each steps very much heat sensitive.
3. Low temperature cause mispairing in annealing.
4. Vent polymerase from Thermus litoralis also may used.

10. Applications of PCR
 Diagnosis of pathogens(slow growing pathogen)
 Diagnosis of specific mutation
 In prenatal diagnosis
 DNA fingerprinting
 In research
 In molecular Archaeology(palaentology )
 Diagnosis of plant pathogens

11. Randomly Amplified Polymorphic
DNA
 Technique used to amplify unknown(random)
DNA segments.
 Williams(1991) develops the technique.
 Type of PCR reaction but segment amplified
by randomly.
 Find the genetic relativeness between two
individuals.
 Previous genetic information is not needed

12. Points to be remembered
Using the discrete DNA profiling principle scientists
Develops three methods.

13. How RAPD works?
Genomic DNA
PCR
amplification
of fragments
elelectrophoresis

14. Steps involved in RAPD technique
Isolation of DNA
Purify the DNA
and run PCR
process
In PCR primer
binds the DNA
randomly
After binding
randomly binded
primer amplify
the template
Amplified
products
separated by gel
electrophoresis
Bands detected by
Ethidium bromide
staining

15. RAPD AP-PCR DAF
Single part of amplification Amplification occurs three parts Two cycle occurs
Normal concentration required(10)
base
High concentration of primer
required(10-50) base
Short concentration of primer
needed(5-8)base
Analyzed by agarose gel Analyzed acrylamide gel Analyzed acrylamide gel
Strained with ethidium bromide
visualized with UV light
Detected by autoradiography Detected with silver straining
---------------------------- ----------------------------------- Complex banding pattern
Comparison between MAAP

16. Advantages of RAPD over RFLP
 No species specific probe required.
 Data collected quickly.
 Analysis of whole genome.
 Small quantity DNA required.
 Does not require blotting or hybridization.

17. Applications of RAPD
 Preparation of genetic map(Arabidopsis, Pines)
 Mapping the genetic trait(barley, maize, pea, rice)
 Fingerprinting
 Tagging of marker

18. Drawbacks of RAPD
• Does not detect allelic variant
• Dominant marker

19. Amplified Fragment Length Polymorphism
 Described by Vos and Zabeau in 1993.
 Combination of RAPD & RFLP technique.
 Superior to RAPD.
 Amplify the same gene from different individual.
 Powerful approach to detect polymorphism .

20. Steps involved in AFLP technique
AFLP
Restriction
digestion
Adaptor
ligation
Amplification
Gel
electrophoresis

21. Individual 1
Individual 2
Digestion of restriction
enzyme
Digestion of restriction
Enzyme(same)
Adaptor binding
Adaptor binding
Steps involved in AFLP technique

22. 1
2
PCR amplification
PCR amplification
Running
on
Agarose
gel
electroph
oresis
Agarose gel electrophoresis
Steps involved in AFLP technique

23. Points to be remembered
 All AFLP marker is dominant.
 Primer should be designed according to adaptor.

24. Advantages & Application
Much batter technique than RAPD.
No need for the known sequence in the genome.
High reproducibility.
Many loci are simultaneously analyzed.