How can I produce milligram quantities of an isotope-labeled DNA oligomer?

Question

I'd like to produce a specific DNA sequence on a milligram-scale and ¹³C¹⁵N-label it. The sequence is around 35 nucleotides long, so chemical synthesis is out due to the exorbitant costs.

I'm also only interested in the single-stranded DNA, so a method that produces double-stranded DNA without any way to easily separate the strands would also not be useful.

What methods are there to produce labelled DNA that fulfill these requirements?

Answer 1

You can also design a plasmid that has your 35mers and raise the bacteria with isotopic carbon and nitrogen C13 acetate and N15 ammonium sulfate will work with E coli in a minimal medium.

this is not cheap, but the cheapest available sources of the isotopes can be used.

Answer 2

I found a couple of methods using M13 phage growing on E coli:

• Reddy P, McKenney K. Improved method for the production of M13 phage and single-stranded DNA for DNA sequencing. Biotechniques. 1996 May;20(5):854-6, 858, 860.

• I Jupin and B Gronenborn. Abundant, easy and reproducible production of single-stranded DNA from phagemids using helper phage-infected competent cells.Nucleic Acids Res. 1995 February 11; 23(3): 535–536.

You could easily adapt these protocols to grow on 13C, 15N-containing medium. Then you could use a restriction enzyme able to cut ss DNA and purify your fragment through a PA gel. Clontech's gigaprep kit (up to 50 mg of DNA) will cost you $546 (in the US, before any quote).

Disclaimer: I've never done any of this!

Answer 3

The cheapest way to make that large quantity of DNA without the need of the whole oligomer being label would be to produce 34 nucleotides by chemical synthesis. The 35 nucleotide can be labeled and ligated to the remaining strands. This should reduce the costs of producing the oligomer since the most expensive stage, the labeling is limited to just the final nucleotide.