By Ahmad Mansour Mohamed Alzohairy Department of Genetics, Zagazig University,Zagazig, Egypt 2009 PCR Techniques

Triple primer RAPD PCR (RAPD stands for Random Amplification of Polymorphic DNA) This is copyrighted material from:-

PCR is used to amplify a known sequence of DNA. Thus, the scientists chooses the sequence they she wants to amplify, then designs primers which will anneal to sequences flanking the sequence of interest.

RAPD reactions are PCR reactions, they amplify segments of DNA which are essentially unknown to the scientist (Random). (Williams et al. 1990)

RAPD primers with an arbitrary sequence. The scientist simply makes up a 10 base pair sequence (or may have a computer randomly generate a 10 bp sequence. Then carries out a PCR reaction and runs an agarose gel to see if any DNA segments were amplified in the presence of the arbitrary primer. (Williams et al. 1990)

Remember! In order for PCR to occur: - The primers must anneal in a particular orientation (such that they point towards each other). - The primers must anneal within a reasonable distance of one another. In this figure which depicts a RAPD reaction, a large fragment of DNA is used the template in a PCR reaction containing many copies of a single arbitrary primer

Finding Differences Between Genomes Using RAPD Analysis If another DNA template (genome) was obtained from a different (yet related) source, there would probably be some differences in the DNA sequence of the two templates. (Williams et al. 1990)

Suppose there was a change in sequence at primer annealing site #2: As shown in this figure, the primer is no longer able to anneal to site #2, and thus the PCR product A is not produced. Only product B is produced.

The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of Primers was further increased by combining two primers in a single PCR. (Klein-Lankhorst et al. 1991)

A new strategy was used to increase RAPD potential in genetic diversity by using three different primers combinations per reaction. Mansour et al. 2008

Mansour et al. 2008

RAPD assay was adapted in mango to direct the amplification of a genome-specific “fingerprint” of DNA fragments (López-Valenzuela et al. 1997; Lowe et al. 2000; Kumar et al. 2001). ISSR was also used extensively for producing molecular markers in horticultural plants such as pear (Pyrus communis) (Corvo et al. 2001), gooseberry (Ribes grossularia subgenus Grossularia) (Lanham and Brennan 1999) and Ribes nigrum L. (Lanham et al. 2000). RAPD & ISSR markers in horticultural

In this investigation, we aimed to provide a comparative assessment by using ISSR and three-primer based RAPD analysis to study genetic diversity among mango cultivars. Thus, we hypothesized that if one primer used in RAPD analysis was not sufficient, we could expect more bands to be detected by using three primers Aim of work

Mansour et al. 2008

A RAPD Triple RAPD Mansour et al. 2008

Mansour et al. 2008

Mansour et al. 2008

Mansour et al. 2008

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