Random amplified polymorphic DNA-RAPD.pptx

Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random.
Williams et al. (1990) developed this technique using very short primers to generate random fragments from template DNAs.
RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.

Principle
The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.
The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.


Procedure
1. DNA is made single stranded by raising the temperature to 940C.
2. In second step, temperature is lowered to 40- 650C which results in annealing of the primer to their target sequences on the template DNA.
3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal.
4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.

5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites.
6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.

1. Random amplified polymorphic DNA Presented by- Nusrat Sheikh

2. Introduction Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random. Williams et a l. ( 1990) developed this technique using very short primers to generate random fragments from template DNAs. RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.

3. Principle The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide . At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.

4. Continued…. The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.

5. Procedure 1. DNA is made single stranded by raising the temperature to 94 C. 2. In second step, temperature is lowered to 40- 65 C which results in annealing of the primer to their target sequences on the template DNA. 3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal. 4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.

6. Continued… 5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites . 6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.

7. RAPD REACTIONS

8. Modifications

9. APPLICATIONS

10. 1. Generating novel specific probes Any diagnostic RAPD marker can be eluted from the gel, reamplified , radiolabelled with 32 P and serve as an inexhaustible supply of probe in Southern analyses. Such probes may be used to exclude the possibility of co-migration of fragments of different sequence but similar size. 2. Analysing mixed genome samples The RAPD technique may be used to generate quantitative estimates of the relative proportions of different genomes in mixed DNA samples.

11. 3. Determination of paternity and kinship relationships By employing fragments that are polymorphic among individuals , RAPD analysis may be used to assess paternity and kinship relationships in large offspring samples . 4. Determination of taxonomic identity By employing different oligonucleotide primers, molecular characters can be generated that are diagnostic at different taxonomic levels . 5. Developing Genetic Markers Linked to a Trait in Question One of the most widely used applications of the RAPD technique is the identification of markers linked to traits of interest without the necessity for mapping the entire genome.

12. Limitations

13. References Random Amplified Polymorphic DNA (RAPD) Markers- Fevzi BARDAKCI Cumhuriyet University, Department of Biology, Sivas-Turkey. Applications of random amplified polymorphic DNA (RAPD ) in molecular ecology- H . Hadrys , M. Balick and B. Schierwater . PCR amplification assays: RAPD .

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