Hello there, hoping you are well,
I have reached a point in my current project where I need to amplify very very short (50mer) non-coding ssDNAs. I understand the options I have available regarding single stranded amplification, but all the resources and tools I have found online for primer design focus on amplifying longer oligonucleotides, especially genes. As I am trying to teach myself this, I wonder if the principles of primer design (i.e. length of 18-24 nts) remain the same for such a short oligo.
Sorry if this is a basic question but I can't get my head around it for some reason haha. Any links to helpful literature would be greatly appreciated.
Many thanks,
Ferg
You can also get the 50bp ssDNA commercially synthesized as a primer.
PCR amplification may result in other biproducts which you may have to remove.
You will have to use Polyacrylamide gel electrophoresis instead of agarose gels followed by gel purification and ethanol precipitation of DNA.
Short sequences and can also be synthesized by primer extension method which utilizes a synthetic ssDNA as template and Klenow fragment (DNA Pol I).
Primer quality depends on the genome present. If amplifying from a huge genome then oligos need to be long to be specific but if the only template is a single sequence then very short primers can be used as there is only one place they can anneal to. Extremely short oligos need a low annealing temperature so can get non specific and also be aware that Taq type enzymes make more errors at low temperatures which is why Sonam Mehrotra suggests using Klenow because the taq is still active even at the annealing temperature but you should be able to increase the amount of ssdna product using asymmetric pcr if the risk of errors near the 3' end of the primer is acceptable
If you do not obtain ssDNA with unknown sequence (for example aptamers or variable locuses), you can get it as a synthesized oligonucleotide where you order your primers. If you want to use PCR, the main principles are the same. But I usually perform the extension step for 15 s. I get short ssDNA with an unknown central region, and I use one biotinylated primer and an ordinary one for an effective strand separation after the PCR.
Yes as Ivan Petushkov says biotin binding works well and another option is to run pcr with one phosphorylated primer then remove one strand with lambda exonuclease
Paul Rutland Sonam Mehrotra that's a good point with the klenow fragment, I overlooked that. Ivan Petushkov fair enough, that reassuring, I was planning on doing asymmetric PCR due to budget constraints but I assume the primers should just be complemetary to the template and that primer length is proportional to template length. Looks like I need to do some more research. Amy Johnson
haha yeah you're right for this project but in the long run it would definitely be cheaper to be able to replicate our oligos indefinitely.- Badges
- Science method