Summary of RAPD method based on Kumar and Gurusubramanian 2011 (Sci Vis 11 (3), 116-124 )

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2. Dr. Ahmed ElFatih ElDoliefy
3/18/2022 2
RAPD-AGERI
PhD, Plant Sciences, Molecular Genetics and Plant Breeding, North Dakota State University (NDSU), Fargo, ND, USA.
MSc. Genetics, Functional Genomics, Ain Shams University (ASU), Cairo, Egypt.
Researcher, Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt.
Source: Kumar and Gurusubramanian 2011

3. 1- Using PCR technique to develop DNA markers
2- A modified PCR technique where single short arbitrary primer recognizes multiple locations throughout
the genomic DNA and produce amplified products with characterized features.
Gene mapping, population genetics, molecular evolutionary genetics, plant and animal breeding.
Fast, cost, efficient, large number of products, no prior knowledge of the genome is required
1- Not suitable for DNA databank
2- Reproducibility
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1-Random priming makes RAPD suitable to compare DNA of biological systems that are not commonly known
Primed by single arbitrary decamer (10 nucleotides) DNA fragments dispersed randomly on a genomic DNA
2- In plant systems where relative few DNA sequences are compared

4. 1- The single-sequenced primer binds to many different loci
2- RAPD products length depend on distance (between binding
primers) and size of template DNA
3- DNA template must have complementary sequences to the
primers
5- Size of RAPD can reach 3.0 kb
6- RAPD is detected on agarose and acrylamide gels
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4- complementation must be in opposite strands, and in opposite
orientation within the distance to be amplified

5. Arbitrary primed-PCR (AP-RAPD)
Primers are 15 bases long with different amplifications and separation conditions.
DNA amplification fingerprinting (DAF)
PCR use shorter primers ≤10 bases
Sequence characterized amplified regions (SCARs) analysis of RAPD polymorphisms
Sequence the RAPD bands after visualization on gel
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1- Each primer directs amplification of several discrete loci (random) in the genome
2- Primers are not binding to single genes out of the whole genome

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Scoring the banding as present (1) or absent (0)
Clear and transparent gels (Fig. 5a)
1) reproducibility—need to repeat experiments
2) thickness
3) size
4) expected segregation observed in a mapping population
1) mismatches at the primer site (must not be reproducible)
2) appearance of a new primer site
3) length of the amplified region between primer sites

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A B C
1 1 1
1 1 1
0 1 1
1 1 1
1 1 1
0 0 1
1 1 1
1 1 1
Software

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Extraction of DNA
DNA must be clean and of high molecular weight
1. Template DNA
*Quality of the template influences the outcome of the PCR
*RNA
*Polymerase inhibitors
*Integrity of the template
*High molecular weight (HMW)
- testing a new template
positive control
negative control
*The recommended amount of template
0.1 ng (Plasmid) to 500 ng (Human)

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2. Primers
10 base primers for PCR
Only 1 primer per reaction
3. Taq DNA polymerase
Thermus aquaticus
Optimal amount
0.5-2.5 U/50 µl reaction volume
Increased enzyme concentration sometimes leads
to decrease specificity
4. MgCl2
Vary from 1-5 mM (1.5 mM when dNTPs are at a
concentration of 20 µM)
High Mg2+ increase non-specificity of primers (background)

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5. dNTPs
Balanced four dNTPs to minimize polymerase error rate
Imbalanced dNTPs mixtures will reduce Taq DNA polymerase fidelity
Increased concentration of dNTP, will reduce free Mg2+, thus interfering with polymerase activity and
decreasing primer annealing
Concentration should be 50-500 µM (each dNTP) and the most commonly used is 200 µM
6. PCR buffer (pH)
(pH 8.3-9.0)

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1. Preparation of master mix for PCR to a PCR tube, add all the ingredients in order
2. Tap the tube for 1-2 seconds to mix the contents thoroughly.
3. Add 25 µl of mineral oil in the tube to avoid evaporation of the contents.
4. Place the tube in the thermocycler block and set the program to get DNA amplification.
5. Carry out the amplification in a thermocycler for 30-40 cycles using the following reaction conditions:

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Does not require DNA sequence information to design specific primers
Quick, simple and efficient
No blotting or hybridization
Small amounts of DNA
Automated
High number of fragments
Arbitrary primers are easily purchased
Unit costs per assay are low compared to other marker technologies
Nearly all RAPD markers are dominant
Enzyme-based reaction
Mismatches
Lack of a prior knowledge on the identity of the amplification products.
Problems with reproducibility
Problems of co-migration

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Genetic diversity/polymorphism
Germplasm characterization
Genetic structure of populations
Domestication
Detection of somaclonal variation
Cultivar identification
Genetics
Plant and animal breeding
Animal-plant-microbe interactions
Pesticide/herbicide resistance
Animal behavior study
Forensic studies
https://www.youtube.com/watch?v=ZW9zPdb_Bs0
http://www.slideshare.net/Cooksinto/chapter-3-recombinant-dna-technology
http://www.powershow.com/view/3b488c-ZjBlZ/Genetic_Engineering_Recombinant_DNA_rDNA_Technology_rDNA_powerpoint_ppt_presentation
https://www.youtube.com/watch?v=XeWW8xfdTEQ
http://www.slideshare.net/mluthfan2/pcr-rapd-dan-rflp?from_action=save

16. Revision
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10- What RAPD stands for?
6- Why RAPD is not adequate for genome databases?
5- What are the basic steps and components of the RAPD?
7- How many primers are binding in a RAPD reaction?
4- The more Mg in RAPD the more products you get-(right?)
1- Mismatching bands in RAPD has nothing to do with dNTPs concentrations-(right?)
9- RAPD is an old technique-(right?)
2- Does the scoring method for RAPD depend on allelism of gene theory?
3- Give example to an advantage and disadvantage of RAPD?
8- Score the RAPD gel in the figure?

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