The Polymerase Chain reaction has wide applications in diagnostic and research laboratories. It is routinely being used in the diagnosis and identification of bacteria. A number of specific primers for the detection of aquatic organisms by PCR have been reported and these primers undergo a long validation process before they are routine in a diagnositc laboratory. For example; the sodB gene of Edwardsiella Tarda was reported by PCR.
The cycling conditions will depend on the type of thermocycler, annealing temperature of the primers, type of Taq enzymes used and if it is a hot-start enzyme or not. A standard set of cycing conditions begins with one cycle at 95 degrees celsius for 5 minutes followed of 25-35 cycles of denaturation at 95 degrees celsius for 30 seconds, annealing at 5-68 degrees celsius at 30 seconds, extension at 72 degrees celsius for 30 seconds. It is suggested that a final cycle should be put in place with an extension time of 10 minutes. The thermocycler can be set to do a hold cycle at 4 degrees celsius.
The primer annealing temperature depends on the length of the primer and the AT/GC concentration. It is recommended that the annealing temperature be set at 5 degrees celsius below the true melting point of the primers. To estimate the annealing temperature, calculate 2 degrees celsius for AT primers and 4 degrees celsius for GC primers. A lower annealing temperature than the optimal annealing temperature may lead to mispriming of non-target sequence or the mis-extension of incorrect nucleotides at the 3' end of the primers.
The optimal number of cycles is between 25-35. Increasing the number of cycles may lead to problems with the PCR. A plateu effect is reached where the product is no longer amplified. As a result, a non-specific product may be amplified preferentially. This plateau is reached according to the number of target copies initially present in the sample and the amount of DNA synthezised. Reagent exhaustion also occurs with an extended number of amplification cycles.
http://www.icampus.ucl.ac.be/courses/SBIM2520/document/genemol/PCR/pcr.jpg
Andika Putra
TG01
6 comments:
Hello Andika,
1)What is a hot-start enzyme?
2)How can increasing the number of cycles lead to problems with the PCR and what type of problems?
3)Why should a final cycle be put in place with an extension time of 10 minutes?
Thankz!
Han Yang
TG01
Hi Andika...for your major project which type of PCR are you using?Real-time PCR?nested PCR?multiplex PCR?Gel based PCR or Reverse Transcriptase PCR?thanks:)
Rachael
Tg01
Han Yang...
A hot-start enzyme is when the sample needs to be pre-heated before PCR
Increasing the number of cycles can cause problems such as; non-specific product may be amplified preferentially.
A final cycle should be put in place to make sure that extension is completed
Rachael..
I will be using gel-based PCR first, then multi-plex PCR for identifying the bacteria. Multi-plex PCR will be done during the final stages of my MP
Hi XD
'A lower annealing temperature than the optimal annealing temperature may lead to mispriming of non-target sequence or the mis-extension of incorrect nucleotides at the 3' end of the primers.'
why is this so?
Tq~
Tan Zhao Rong
tg01
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