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real_time_pcr [2012/04/16 12:24]
anniearchambault
real_time_pcr [2012/04/16 12:25]
anniearchambault
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 Using double stranded DNA binding dyes, SYBR green I or SYBR green II for instance, to detect the amount of amplified DNA in real time is the most commonly used method in biodiversity science. Main advantages are that it is easy to set up and convenient, since the user has only to optimize forward and reverse primers sequences and reaction conditions. Limitations are that the double stranded DNA binding dyes will also anneal to nonspecific amplified products and primers dimers. ​ Using double stranded DNA binding dyes, SYBR green I or SYBR green II for instance, to detect the amount of amplified DNA in real time is the most commonly used method in biodiversity science. Main advantages are that it is easy to set up and convenient, since the user has only to optimize forward and reverse primers sequences and reaction conditions. Limitations are that the double stranded DNA binding dyes will also anneal to nonspecific amplified products and primers dimers. ​
  
-Sequence specific fluorescent probes (TaqMan probes) anneal to the target DNA fragment and will emit their fluorescence when degraded by the 5' to 3' exonuclease activity of the polymerase. The Wikipedia page [[wp>http://​en.wikipedia.org/​wiki/​TaqMan]] explains in more detail the process. This method has the advantage of increased specificity,​ since the probe will not anneal to nonspecific amplified products and primers-dimers. It also enables the multiplexing of up to four or five reactions in a single tube, when using probes attached to different fluorescent molecules. The method is however more complex to set up, as the user will need to optimize the sequence and reaction conditions of the specific probe in combination with specific amplification primers. Moreover, because the probe is sequence specific, it cannot be transferred to another experiment. ​+Sequence specific fluorescent probes (TaqMan probes) anneal to the target DNA fragment and will emit their fluorescence when degraded by the 5' to 3' exonuclease activity of the polymerase. The Wikipedia page [[wp>​TaqMan]] explains in more detail the process. This method has the advantage of increased specificity,​ since the probe will not anneal to nonspecific amplified products and primers-dimers. It also enables the multiplexing of up to four or five reactions in a single tube, when using probes attached to different fluorescent molecules. The method is however more complex to set up, as the user will need to optimize the sequence and reaction conditions of the specific probe in combination with specific amplification primers. Moreover, because the probe is sequence specific, it cannot be transferred to another experiment. ​