I have been trying this since a year by now. I need three different sizes of single stranded DNA. The shortest one is easily available as purchased oligos, however the other two, 280nt and 360nt long are too long it seems to be synthesized artificially.
Are there any companies that synthesize such long oligos? I have been trying various techniques like Late-PCR, denaturing with one strand biotinylated and a lot more to create one by myself. But it doesn't seem to work. Can I get some suggestions on creating single stranded DNA of 280nt and 360nt respectively? What else can I try?
Hi, I can think of 3 methods.
1) Ligation. I think it is possible to ligate several shorter pieces. For example, you can take four 70-mers, bridge them by three short complementary strands that bridge across DNAs. Then you can use an enzyme like T4 ligase to repair the nick. Your single stranded DNA can be recovered by running gels such as denaturing gel. You can ligate fewer pieces, but you have to remember that longer the (synthetic) DNA, more chances for error. (http://www.sciencemag.org/content/337/6098/1097/suppl/DC1)
2) Rolling replication scheme. This can yield a ssDNA that is much longer than what you want, but this may work for you.
http://pubs.rsc.org/en/Content/ArticleLanding/2011/SM/C1SM05297G#!divAbstract
http://www.ncbi.nlm.nih.gov/pubmed/24792418
3) Gene synthesis. Companies such as Integrated DNA Tech makes short gene fragments (gBlocks). If you can find a way to denature and separate the two, this will work.
I hope this helps.
http://eu.idtdna.com/pages/products/genes/gblocks-gene-fragments
Thank you for your answer Yuji. I cannot go for the ligation method. I did think of it earlier, however, the technique would give a very low yield overall. So I decided not to go for it. The rolling replication scheme seems interesting.I haven't done this before and I feel that its worth trying. Thanks a lot. The info was of great help.
- Badges
- Science topic