Difficult question, but we're trying to carry out a PCR that produces a single stranded amplicon. We've tried using only the forward primer or the forward to reverse in a 500:1 ratio, without much success.
Does anyone have any tips or suggestions?
Ok, so first I'd make a purified PCR product (with fwd and rev primers), because if you use genomic DNA as a template there's nothing to stop a single primer being extended indefinitely: rather than a population of uniform single strand products you'd have a whole load of mixed length products. If you use a PCR fragment as template then the polymerase will just fall off when it reaches the end, so all your amplicons will be the same size.
Thinking about it, I would probably advise using LOTS of your starting PCR fragment as template: given you're only going to be increasing your product linearly. How much would have to determined empirically, but polymerases can take quite a lot, and saturating the enzyme with the correct template is better than undershooting, really. I'm assuming you're doing this to make a lot of single stranded amplicon, not to do anything quantitative, incidentally.
If you start with a lot of DNA, you could probably use fewer cycles, but I'd still use a lot. 64 cycles of linear PCR will only produce as much DNA as maybe 6 cycles of exponential PCR, so yeah: aim high. It's not like the DNA will degrade, so again, overshooting would be better than undershooting.
Of course, depending on how long your amplicon is, it may be cheaper and easier to just get it synthesized. Less fun, admittedly.
(or there's this old-school method, which I believe a lot of probe labelling kits use: attached)
How many cycles are you doing? Remember that single-primer PCR isn't exponential (less PCR, more just..PR), so rather than 35 cycles you may need several hundred for a decent yield.
I would assume you'd want to prepare a decent quantity of purified double-stranded template, then use that as your starting material for a PCR with your forward primer alone and do many, many many many cycles. Then either separate single stranded population from double stranded via gel electrophoresis, or use a dsDNA specific nuclease.
Admittedly I have only been doing 45 cycles. I've got human cancer cell line derived DNA so I have shed loads to put in.
I worry about saturating the enzyme if I put more than I usually put in (say 200ng) how much input would you usually use?
And final question (sorry) - how much is "many many cycles"? 200? 300?
Thanks
Andrew
Ok, so first I'd make a purified PCR product (with fwd and rev primers), because if you use genomic DNA as a template there's nothing to stop a single primer being extended indefinitely: rather than a population of uniform single strand products you'd have a whole load of mixed length products. If you use a PCR fragment as template then the polymerase will just fall off when it reaches the end, so all your amplicons will be the same size.
Thinking about it, I would probably advise using LOTS of your starting PCR fragment as template: given you're only going to be increasing your product linearly. How much would have to determined empirically, but polymerases can take quite a lot, and saturating the enzyme with the correct template is better than undershooting, really. I'm assuming you're doing this to make a lot of single stranded amplicon, not to do anything quantitative, incidentally.
If you start with a lot of DNA, you could probably use fewer cycles, but I'd still use a lot. 64 cycles of linear PCR will only produce as much DNA as maybe 6 cycles of exponential PCR, so yeah: aim high. It's not like the DNA will degrade, so again, overshooting would be better than undershooting.
Of course, depending on how long your amplicon is, it may be cheaper and easier to just get it synthesized. Less fun, admittedly.
(or there's this old-school method, which I believe a lot of probe labelling kits use: attached)
I know it probably sounds naive, but why don't you use a biotinylated primer for the strand that you are interested in, do a normal PCR, denature and dilute the product and then capture the correct strand? I am sure biotin-streptavidin interaction can withstand formamide, salt and temperature that DNA-DNA base pairing cannot.
Just a thought.
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