Q&A in AGH635 Course: “How To Do Primer Screening in RAPD Marker Analysis for Genetic Diversity?”

Still related on how to go ahead in RAPD marker analysis, the second step is “Primer Screening.” Along in this topic, one of my AGH635. Anmolsel course participant asked me the following question: “How To Do Primer Screening in RAPD Marker Analysis for Genetic Diversity?” Here is my answer to such a question.

The purpose of doing primer screening is to save some research resources. Let’s consider the following: you have samples of 200 random primers to use in RAPD markers analysis. Under your research, you want to analyze genetic distance and diversity of 100 samples. You have a choice of either (1) applying all primers to all samples, or (2) doing primer screening, select primers generating informative markers, and genotype the samples with selected primers. The cost differences between the two are outline in the following table (Table 1).

 Table 1. Cost comparison of analyzing 100 individuals using RAPD markers: either incorporate primer screening or without one

Table 1. Cost comparison of analyzing 100 individuals using RAPD markers: either incorporate primer screening or without one

If without primer screening, it will need a minimum of 20,000 PCR reaction (200 primers x 100 individual=20,000). In this case, it resulted in the same amount of data as it will be generated by incorporating primer screening (data for markers generated by 75 informative primers)

  • However, if it was conducted by incorporating primer screening, the required Rxt : (1) 200 primers are tested using 5 array of selected samples to find informative markers (use steps below; sub-total PCR Rxt. 1,000 ). Only primers generating informative markers are selected (let’s assume only 75 primers generate informative markers (sub-total PCR Rxt. 7,500). Therefore, the total PCR Rxt used in the analysis is only 8,500. One can save (i.e. 11,500 of PCR Rxt). some of the resources that can be used for other activities.
  • Since there is no guarantee which of the random primers will generate informative marker, it is always advisable to screen the most informative primers and only use these primers for genotyping the whole members of the population. The objective of primer screening is to identify such informative primers.

Steps for primer screening in RAPD marker analysis for the purpose of genetic diversity studies include :

  • Select five samples, use ones showing as diverse phenotypes as possible, from the studied population and use them as the samples for primer screening.
  • Select some random oligo-nucleotide primers to be tested. In this case, if you have already had prior knowledge about oligonucleotide primer that are polymorphic (i.e. the target primers could be identified from published data of the same crops or related crop you investigate), then test those primers first. If you do not have prior knowledge about the primers, then you should do primer screening yourself from scratch.
  • The number of random oligo-nucleotide primers one should screen in this step depend on the total number of markers (i.e. the number of loci) that one would analyze in their research activity and the number informative markers generated per primer.
  • Just as a rule of thumbs about what the total number of loci should be analyzed, if the chromosome numbers of the studied crop are 16, it is always good to select the number of loci at least 2-3 times the number of chromosomes. Hence, one should analyze a total of at least 32-48 polymorphic loci. If each primer is assumed to produce at least 2 polymorphic RAPD marker loci, it should need at least 16-24 primers.
  • To get 16-24 primers, each producing at least 2 polymorphic markers, one should not just screen 16-24 (i.e. ones should screen more than that, and look for the best one) since some of the tested primers: either (1) might not produce amplified product at all, (2) even if it produces amplified product, it may only produce one marker per primer, or (3) even if it produce more than one amplified product, they may not be polymorphic. Tested primers producing such amplified products (either point [1], [2] or [3]) may be undesirable or useless for the genetic diversity analysis.
  • Rule of thumb in selecting the primers are as follows: select primers that when use to amplify 5 selected samples, at least: (1) the tested primers generate amplified PCR product (i.e. generate RAPD marker), (2) the tested primers generate two or more markers per primer (the more the better), (3) the tested primers generate clear and clearly spaced markers (i.e. would be scored easily), and (4) the tested primers generate as many polymorphic markers as possible.
  • Once the selected primers are identified, they can be used to genotype all the samples in the studied populations.

About PMB Lab: Prof. Sudarsono

This blog is dedicated as a communication media among alumni associated with PMB Lab, Dept. of Agronomy and Horticulture, Fac. of Agriculture, IPB, Bogor – Indonesia. It contains various information related to alumni activities, PMB Lab’s on going activities and other related matters.
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