about "LAM-PCR" technique - ask for help (Nov/11/2005 )

Dear All,

Is there anyone know the "LAM-PCR" method? My question is that how to label the 5'-biotinylated primer, is there any kit available.

Thousands thanks!

Jane

hi
i suppose biolynilated primer itself is labelled. please be specific in your question.

you can synthesise primers with 5' biotynilation modification. maybe termonal transferase can also achieve this with biotin nucleotide

I once ordered a primer labelled with biotin. It didn't work, possibly due to steric hindrance, but I'd talk with your oligo synthesis company

Hi, LAM PCR technique enables one to sequence an unknown region. It has nothing to do with biotinylated primers. This is a patented method of LAM-PCR (linear amplification-mediated PCR) allows the amplification and sequencing of unknown regions in every given genome, no matter if from a bacterium, plant, animal or human. You need to send only 5 µg of purified genomic DNA and the sequence of a known DNA segment. There is a company called GATC biotech which offers this service, i doubt if u can carry out this procedure in your lab.


Otherwise biotin end labeled primers are used if u r detecting pcr products by elisa.....u then use avidin coated wells to capture the pcr products which have biotin ends.

hope this helps.
good luck.
microbug2k

Dear ALL,

Thank you so much for your information. It really helps. I have heard the company called GATC biotech about the service, but I think it is better to set up this method in our lab. Do you think it could make LAM-PCR works in our lab if I buy 5' biotynilation primer and also the GenomeWalker kit?

Thanks!

Jane

There are lots of techniques for PCR walking into unknown regions from known regions. My personal favorite is panhandle - see this thread for more details

genome sequencing

Daniel

DNA sequencing protocols