Hi,

hopefully I have understand your problem.

I think you can tag one primer of your first PCR with the oligo and label this primer with biotin- than you bind the PCR product onto streptavidin beads and wash away the second DNA strand. The result should be a tagged ssDNA.

You could try to put a DNA oligo on the 3'OH of ssDNA using " T4 RNA ligase 1 " (New England Biolabs - Cat no. M0204S ). Note that the oligo should be 5' phosphoryl-terminated (this is required for ligation to the 3' OH of the target ssDNA). It is better to use primer with lacks 3' OH to prevent generation of the primers oligomers.

THanks Norman.. actually I want to do PCR using a known primer, and a ligated primer to the 3' end of an unknown sequence.

Hi Eugene, I think that should work but I'm not sure if I will have to purify the ssDNA using Normans suggestion. If there is a lot of dsDNA around, it may interfere with the labeling. So maybe this is a two step process.

Hi Eugene, You can tail ssDNA with Terminal transferase (TdT). You can find the protocol to do that in 5'RACE pcr from Invitrogen. It works very well

I would try without purification, understand that you will have an excess of ssRNA product of PCR run off reaction. Also, make sure that for amplification of the product after primer ligation you will not use the same primer which was used in run off reactions, chose a region downstream in the run off product.

Another suggestion, if all you have to do is to identify positions of junctions, try inverted PCR, this approach is widely used to identify insertion sites in inserted mutagenesis, see for example :

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1803754/

"...Isolation of Target Sites. To amplify the junctions of Ds and upstream flanking plant DNA, inverse PCR was carried out using PstI, XbaI, and AsnI digests (2 μg). After 1 h of DNA circularization, DNA ligase treatment, and removal of salts using the QIAquick PCR purification kit (Qiagen), DNA digests were used as templates for PCR (30 cycles) with primers Pr17 and Pr18. The amplified products (1 μL) were used as a template for subsequent PCR (35 cycles) with a set of nested primers (Pr19 and Pr20). The Ds junctions with downstream flanking DNA were obtained by using EcoRI, AflIII, and EcoRV digests with Pr21-Pr22 for the first PCR, and Pr23-Pr24 for the second PCR. DNA fragments were separated by agarose gel electrophoresis, isolated using Qiagen's gel isolation kit, cloned into plasmid pGEMT-Easy (Invitrogen), and sequence analyzed using Big Dye Terminator, ....".

(of course you should choose restriction enzymes which are the best for your DNA nucleotide composition etc..

cheers

E

would this allow me to poly A tail the ssDNA and then use RT with oligodT to make dSDNA?

Hi Rick,

I did not read your question carefully. I think you should try T4 RNA Ligase 1 or Terminal Transferase as suggested by Eugene and Marta

Hi Rick

In addition to the above suggestions, you can also use Poly A polymerase or Poly U polymerase enzymes for 3' tailing and ..this will also enable you to do reverse transcription using oligodT..

bye

Poly A Polymerase do not work with deolxynucleotides (DNA). You can use the T4RNA ligase with dATP to get polyAtail at 3' end. Or other option to go for T4 RNA ligase base 5'P-oligos or TdT.