Why do you want to increase the specificity in RAPD? Usually RAPD is done with 37 or 42 degree C as annealing temperature. Touch down PCR is done for gene-specific amplification to increase the stringency.
Could you tell us if the reason you are inquiring about the use touchdown is to be able to figure out a temperature that would generate a RAPDs banding pattern (or a specific band) that you have obtained previously in a consistent manner?
Touchdown has been proposed as a way to improve reliability of RAPD, so you can obtain the same pattern even when using different thermal cyclers.
I haven't used touchdown per se, only 4 initial cycles at a higher annealing temperature followed by 36-40 cycles at a lower temperature. However, you can check touchdown programs for RAPD and the results obtained here: http://www.biotechniques.com/multimedia/archive/00009/97234bm27_9762a.pdf
We have used touch down for the purpose described by Gustavo, but with limited success in terms of pattern repeatability with one exception detailed below. In the process of optimizing RAPDs, we found that the two most important factors determining repeatability were 1) template quality and quantity, 2) Taq polymerase brand and buffer system. We tried about 8 different Taqs from different vendors and formulations, obtaining very poor results in terms of repeatability of RAPDs. The single Taq that was consistent in generating repeatable patterns, inclusive when altering annealing temperature was AccuStart Hi-Fi Taq (Quanta Biosciences, Gaithersburg MD). Thats said, template quality and quantity are a very strong factor determining RAPD patterns. We were consistently obtaining 'diagnostic' differences between samples, until we noticed that DNA source (tissue type) and consequently the quality and quantity were the factors explaining the results.
Thanks very much 4 all of you. Thanks a lot Gustavo Acevedo-Hernandez for the valuable link you shared. I will try it and hope to get a good result as they did. However, i really amazed of the concentration of the primer they used. isn't it very high? I though of typing mistake.
Thanks Jaime Alvarado Bremer , i agree with you. actually i obtained completely different result with the same sample when using different quantities of the template DNA.
Why I want to use a touchdown PCR for RAPD? as being answered by Gustavo Acevedo-Hernandez. to obtain a more reliable result (to improve the reliability of the technique.
Now that you mention it Marwa, I think you are right about the primer concentration in that report. I tipically use 400 nM of primer, and I haven't seen much higher concentrations in other protocols. I would think it is a typo and it should say 200 nM... just my guess (?).
Yes, it should be 200 nM. i have used 600 nM. the recommended/ allowed concentration of the primer is (100 nM - 600 nM) as i found in literature.
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