Sequencing of scarce real-time amplicons - (Jun/18/2009 )

Dear colleagues,

does anyone have experience on a succesful sequencing of a scarce Taqman real-time PCR product? How have you managed to do that?

I have a very little amount (Ct-value 30-35; primary volume 25 ul) of a small (88/89 bp) amplicon, that is very challenging to sequence. Almost all of the original sample RNA has already been used for different attempts. Unfortunately the sensitivities of my conventional PCRs are insufficient, so I am not able to amplify my target with them. I have tried
1) Qiagen PCR purification kit
2) Qiagen gel extraction kit
3) TA-cloning and
4) ExoSAP purification,
all followed by sequencing attempt with the real-time primers that gave the positive result. I have also tried to amplify the amplicon with the real-time primers but with conventional method to get more template; this did not function.

What could I try next? It would be utmost important to find the sequence since this is the first finding of a virus in our country...

Did you try to sequence your TA-cloned taqman product using the CLONING PRIMERS? (such as PUCM13 etc.) These primers would ensure your product is full length when sequenced. Gel purify your PCR products, making sure the kit you use does not wash away <100bp products. SOme kits do that. Then directly clone into TA vectors. Select positive clones using the universal primers then send the whole purified plasmid for sequencing with the same universal primers.

ACtually what do you mean when you say you cannot get a sequencing result?

Also, where did the Taqman sequence come from? If it is so specific, then you do not need sequencing because the Taqman is very specific. The product detected is the sequence that you want

Hope this helps.

Chris