suitable length DNA for PCR? - (Feb/05/2008 )
Hi folks,
I have a dumb question about PCR. What is the suitable length range of DNA for PCR reaction? What is the maximum length of DNA PCR can produce? Thanks.
-ionchannelbk-
It depends very much on what kind of polymerase you are using and what you want to do with the PCR product.
For a polymerase there 3 aspects to it, processivity, speed, error rate and cost
- Processivity is the average number of nucleotide a polymerase will add before it falls off. Low processivity and the polymerase falls off quickly before adding too many base pairs.
- Speed is how many base pair said polymerase will add per second
- And error rate is how many mistakes are made for a given number of basepairs added.
- Cost is the amount of money you can use before the boss comes looking for you.
We polymerases such as simple basic Taq which are cheap, but rather slow with high error rate and low processivity.
Taq is good for around 500bp. Anything above 1kb and you will have to start optimising condition. Which for some application like colony PCR is not possible. And by 3kb or so nearly every single DNA molecule will have at least one mistake. Useful for cloning only very short fragments.
On the other extreme you have polymerase such as Phusion, fast, very high processivity, rather low error rate and quite expensive. Works well to 20kb and above. But too expensive to use for anything but long range PCR.
In the middle you have polymerase such as Vent and KOD-hifi. Amplification range with 1kb to 5kb. The common workhorse for cloning in my lab.
As for maximum lenghts; The longest I have read about is 50kb. The longest I have seen done is ~20kb. And the longest I have done is ~10kb.
-perneseblue-
QUOTE (perneseblue @ Feb 5 2008, 02:55 PM)
It depends very much on what kind of polymerase you are using and what you want to do with the PCR product.
For a polymerase there 3 aspects to it, processivity, speed, error rate and cost
Taq is good for around 500bp. Anything above 1kb and you will have to start optimising condition. Which for some application like colony PCR is not possible. And by 3kb or so nearly every single DNA molecule will have at least one mistake. Useful for cloning only very short fragments.
On the other extreme you have polymerase such as Phusion, fast, very high processivity, rather low error rate and quite expensive. Works well to 20kb and above. But too expensive to use for anything but long range PCR.
In the middle you have polymerase such as Vent and KOD-hifi. Amplification range with 1kb to 5kb. The common workhorse for cloning in my lab.
As for maximum lenghts; The longest I have read about is 50kb. The longest I have seen done is ~20kb. And the longest I have done is ~10kb.
For a polymerase there 3 aspects to it, processivity, speed, error rate and cost
- Processivity is the average number of nucleotide a polymerase will add before it falls off. Low processivity and the polymerase falls off quickly before adding too many base pairs.
- Speed is how many base pair said polymerase will add per second
- And error rate is how many mistakes are made for a given number of basepairs added.
- Cost is the amount of money you can use before the boss comes looking for you.
Taq is good for around 500bp. Anything above 1kb and you will have to start optimising condition. Which for some application like colony PCR is not possible. And by 3kb or so nearly every single DNA molecule will have at least one mistake. Useful for cloning only very short fragments.
On the other extreme you have polymerase such as Phusion, fast, very high processivity, rather low error rate and quite expensive. Works well to 20kb and above. But too expensive to use for anything but long range PCR.
In the middle you have polymerase such as Vent and KOD-hifi. Amplification range with 1kb to 5kb. The common workhorse for cloning in my lab.
As for maximum lenghts; The longest I have read about is 50kb. The longest I have seen done is ~20kb. And the longest I have done is ~10kb.
Thank you, perneseblue. Your reply is very informative.
-ionchannelbk-