Nucleic acid lateral flow (NALFIA) - (Jan/05/2011 )

Anyone knows where I can find informations about NALFIA? I'm starting in this area now, but I'm having troubles to find protocols or articles about that.

Does anyone know anything about it and help me?

Thank you!

angellica on Wed Jan 5 12:00:25 2011 said:

Hi! Thank you for your answer!

I'm doing a technique also called DNA strip, oligo strip or NALFIA.
The technique is done as follows:

DNA is extract using a kit from Sigma (Extract-N-Amp) and then amplified by PCR using primers tagged.
A primer is labeled at the 5 'biotinylated and the other is labeled with FITC, also in 5 '.
Thereafter, the detection of amplified samples is done using the immunochromatographic technique. A nitrocellulose membrane is impregnated as follows:
- The test zone is impregnated with anit-FITC
-A control zone with biotin
-The conjugate is composed of streptavidin conjugated with colloidal gold.

It is placed 10 ul 50ul of the sample and running buffer and the reaction ocurred for 5 minutes.

If the sample is positive form a band in the test region and control region, and if negative will form a band only in the test region.

The problem is that once you get the amplified sample (and checked on agarose gel) there is a problem in the detection of the sample by nitrocellulose membrane.
We could only detect the sample after purifying the PCR product and yet with a weak signal at a concentration of 10 ul. When we increased the amount of sample to 20 ul test occurs disappearance of the band.

We believe in a possible steric hindrance...

Does anyone know anything about it?

Thanks for your help!

Hello,

Sorry it has taken me so long to reply, but I did not check this thread since there was not a response in over 2 months from when I last posted. I thought you had figured it out. Maybe you have by now??

Regards